Methods for determining resistance or susceptibility to HIV entry inhibitors

ABSTRACT

The invention provides a method for determining whether a human immunodeficiency virus is likely to be more resistant to a viral entry inhibitor than a reference HIV. In certain aspects, the methods comprise comparing the length of one or more variable regions of an envelope protein of the HIV or a number of glycosylation sites on the envelope protein of the HIV to a length of one or more corresponding variable regions of an envelope protein of the reference HIV or a number of glycosylation sites on the envelope protein of the reference HIV, wherein the HIV is likely to be more resistant to the CD4 binding site entry inhibitor than the reference HIV when the HIV has longer variable regions than the reference HIV or the HIV has more glycosylation sites than the reference HIV.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/921,751 (issued as U.S. Pat. No. 9,506,121) filed Dec. 4, 2007,which is a § 371 national phase application of PCT Application No.PCT/US2006/022071 filed Jun. 6, 2006 (expired), which claims priority toU.S. Provisional Application No. 60/765,333 filed Feb. 4, 2006 and U.S.Provisional Application No. 60/688,170 filed Jun. 6, 2005. The contentsof each of these applications are hereby incorporated by reference intheir entirety.

Throughout this application, various publications are referenced byauthor and date within the text. Full citations for these publicationsmay be found listed alphabetically at the end of the specificationimmediately preceding the claims. All patents, patent applications andpublications cited herein are hereby incorporated by reference in theirentirety. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art as known to those skilled therein asof the date of the invention described and claimed herein.

2. BACKGROUND

Enveloped animal viruses attach to and enter the host cell via theinteraction of viral proteins in the virion membrane (envelope proteins)and cell surface proteins (virus receptors). Receptor recognition andbinding are mediated by the surface envelope protein. Virus entry is anattractive target for anti-viral treatment; numerous drugs that aredesigned to block virus attachment or membrane fusion have been or arecurrently being evaluated in preclinical or clinical studies (Richman,1998; PhRMA, 1999; Stephenson, 1999). For example, the attachmentinhibitor SCH-D, which blocks the interaction between viral membraneproteins and CCR5, is currently being evaluated in clinical studies forits effectiveness as an anti-viral treatment (Shurman, 2004). Otherentry inhibitors currently under investigation include UK-427857(Pfizer), TNX-355 (Tanox Inc.), AMD-070 (AnorMED), Pro 140 (Progenics),FP-21399 (EMD Lexigen), BMS-488043 (Bristol-Myers Squibb), andGSK-873,140 (GlaxoSmithKline). One entry inhibitor, T-20(Roche/Trimeris), has been approved for treatment of HIV infection bythe United States Food and Drug Administration.

As these drugs continue to be developed and enter the clinic, assays areneeded that can rapidly and easily detect the emergence of viruses withreduced susceptibility to entry inhibitors. In particular, methods fordetermining whether an HIV is resistant to an entry inhibitor, e.g.,PRO542, TNX-355, monoclonal antibody B4, monoclonal antibody B12, etc.,are needed. These and other unmet needs are provided by the presentinvention.

3. SUMMARY

In certain aspects, the invention provides a method for determiningwhether an human immunodeficiency virus (“HIV”) is likely to be moreresistant to an HIV entry inhibitor than a reference virus. In certainaspects, the invention provides a method for determining whether an HIVis likely to be more resistant to a CD4 binding site entry inhibitorthan a reference HIV, comprising comparing the length of one or morevariable regions of an envelope protein of the HIV or the number ofglycosylation sites on the envelope protein of the HIV to the length ofone or more corresponding variable regions of an envelope protein of thereference HIV or the number of glycosylation sites on the envelopeprotein of the reference HIV, respectively, wherein the HIV is likely tobe more resistant to the CD4 binding site entry inhibitor than thereference HIV when the HIV has a longer variable region or regions thanthe reference HIV and/or the HIV has more glycosylation sites than thereference HIV. In certain embodiments, the CD4 binding site entryinhibitor is selected from the group consisting of PRO542, TNX-355 andmonoclonal antibody B12. In certain embodiments, the reference HIV isNL4-3, HXB2, or SF2. In certain embodiments, the HIV has a longervariable region or regions than the reference HIV. In certainembodiments, the HIV has more glycosylation sites than the referenceHIV. In certain embodiments, the HIV has a longer variable region orregions and more glycosylation sites than the reference HIV.

In another aspect, the invention provides a method for determiningwhether an HIV is likely to be more resistant to a CD4-blocking entryinhibitor than a reference HIV, comprising comparing the length of oneor more variable regions of an envelope protein of the HIV or the numberof glycosylation sites on the envelope protein of the HIV to the lengthof one or more corresponding variable regions of an envelope protein ofthe reference HIV or the number of glycosylation sites on the envelopeprotein of the reference HIV, respectively, wherein the HIV is likely tobe more resistant to the CD4 binding site entry inhibitor than thereference HIV when the HIV has shorter variable regions than thereference HIV and/or the HIV has fewer glycosylation sites than thereference HIV. In certain embodiments, the CD4-blocking entry inhibitoris monoclonal antibody B4. In certain embodiments, the reference HIV isNL4-3, HXB2, or SF2. In certain embodiments, the HIV has a shortervariable region or regions than the reference HIV. In certainembodiments, the HIV has fewer glycosylation sites than the referenceHIV. In certain embodiments, the HIV has shorter variable regions andfewer glycosylation sites than the reference HIV.

In another aspect, the invention provides a method for determiningwhether an HIV is likely to exhibit altered susceptibility to an entryinhibitor, comprising detecting, in a nucleic acid encoding an envelopeprotein of the HIV, a mutation in a codon corresponding to codon 261 ofreference HIV strain HXB2, wherein the presence of a mutation in codon261 indicates that the HIV is likely to be resistant to the entryinhibitor.

In another aspect, the invention provides a method for determiningwhether an HIV is likely to exhibit altered susceptibility to an entryinhibitor, comprising detecting, in a nucleic acid encoding an envelopeprotein of the HIV, a mutation in a codon corresponding to codon 117 andat codon 421 of reference HIV strain HXB2, wherein the presence of themutations indicates that the HIV is likely to be resistant to the entryinhibitor.

In another aspect, the invention provides a method for determiningwhether an HIV is likely to exhibit altered susceptibility to an entryinhibitor, comprising detecting, in a nucleic acid encoding an envelopeprotein of the HIV, a mutation in a codon corresponding to codon 121 orcodon 298 reference HIV strain HXB2, wherein the presence of themutations indicates that the HIV is likely to be resistant to the entryinhibitor.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: Structure of Envelope Expression and Viral Expression Vectors.

The HIV envelope expression vector (pHIVenv) is modified to acceptenvelope sequences that have been amplified from subject plasma samples.The designations a/b and c/d, refer to restriction endonuclease sitespositioned at the 5′ and 3′ end of the HIV-1 envelope polyprotein(gp160). The HIV expression vector (pHIVlucΔU3) encodes all HIV proteinsexcept the envelope polyprotein. A portion of the envelope gene has beendeleted to accommodate an indicator gene cassette, in this case, fireflyluciferase, that is used to monitor the ability of the virus toreplicate in the presence or absence of anti-viral drugs. The 3′ U3region has been partially deleted to prevent transcription from the 5′LTR in infected cells. Virus produced in this system is limited to asingle round of replication.

FIG. 1B: Cell Based Entry Assay

In this embodiment, drug susceptibility, co-receptor tropism and virusneutralization testing are performed by co-transfecting a host cell withpHIVenv and pHIVlucΔU3. The host cell produces HIV particles that arepseudo-typed with HIV envelope sequences derived from the test virus orsubject sample. Virus particles are collected (˜48 h) after transfectionand are used to infect target cells that express HIV receptors (e.g.CD4) and co-receptors (e.g. CXCR4, CCR5). After infection (˜72 h) thetarget cells are lysed and luciferase activity is measured. HIV mustcomplete one round of replication to successfully infect the target hostcell and produce luciferase activity. If the virus is unable to enterthe target cell, luciferase activity is diminished. This system can beused to evaluate susceptibility to entry inhibitors, receptor andco-receptor tropism, and virus neutralization.

FIG. 2A and FIG. 2B: HIV Envelope Expression Vectors.

HIV envelope sequences are amplified from subject samples and insertedinto expression vectors using restriction endonuclease sites (5′ a/b and3′c/d). Envelope transcription is driven by the immediate early genepromoter of human cytomegalovirus (CMV). Envelope RNA is polyadenylatedusing an simian virus 40 (SV40) polyadenylation signal sequence (A+). Anintron located between the CMV promoter and the HIV envelope sequencesis designed to increase envelope mRNA levels in transfected cells.FL-express full-length envelope proteins (gp120, gp41); ΔCT-expressenvelope proteins (gp120, gp21) lacking the C-terminal cytoplasmic taildomain of gp41; +CT-express envelope proteins (gp120, gp41) containing aconstant pre-defined gp41 cytoplasmic tail domain; gp120-express gp120proteins derived from the subject together with a constant pre-definedgp41; and gp41-express a constant pre-defined gp120 together with gp41proteins derived from the subject.

FIG. 3A: Co-Receptor Tropism Screening Assay.

In this figure, the assay is performed using two cell lines. One cellline expresses CD4 and CCR5 (top six panels). The other cell lineexpresses CD4 and CXCR4 (bottom six panels). The assay is performed byinfecting cells with a large number of recombinant virus stocks derivedfrom cells transfected with pHIVenv and pHIVlucΔU3 vectors. The exampleshown represents the analysis of 96 viruses formatted in a 96 well plateinfections are performed in the absence of drug (no drug), or in thepresence of a drug that preferentially inhibits either R5 tropic (CCRinhibitor) or X4 tropic (CXCR4 inhibitor) viruses. Co-receptor tropismis assessed by comparing the amount of luciferase activity produced ineach cell type, both in the presence and absence of drug (see FIG. 3Bfor interpretation of assay results).

FIG. 3B: Determining Co-Receptor Tropism.

In this figure, the results of the assay are interpreted by comparingthe ability of each sample virus to infect (produce luciferase activity)in cells expressing CD4/CCR5 (R5 cells) or cells expressing CD4/CXCR4(X4 cells). The ability of a CCR5 or CXCR4 inhibitor to specificallyblock infection (inhibit luciferase activity) is also evaluated. X4tropic viruses infect X4 cells but not R5 cells. Infection of X4 cellsis blocked by the CXCR4 inhibitor. R5 tropic viruses infect R5 cells butnot X4 cells. Infection of R5 cells is blocked by the CCR5 inhibitor.Dual tropic or X4/R5 mixtures infect X4 and R5 cells. Infection of R5cells is blocked by the CCR5 inhibitor and infection of X4 cells isblocked by the CXCR4 inhibitor. Non-viable viruses do not replicate ineither X4 or R5 cells.

FIG. 4A: Measuring Entry Inhibitor susceptibility: Fusion Inhibitor.

In this figure, susceptibility to the fusion inhibitor T-20 isdemonstrated. Cells expressing CD4, CCR5 and CXCR4 were infected in theabsence of T-20 and over a wide range of T-20 concentrations α-axis log10 scale). The percent inhibition of viral replication (y-axis) wasdetermined by comparing the amount of luciferase produced in infectedcells in the presence of T-20 to the amount of luciferase produced inthe absence of T-20. R5 tropic, X4 tropic and dual tropic viruses weretested. Drug susceptibility is quantified by determining theconcentration of T-20 required to inhibit 50% of viral replication(IC₅₀, shown as vertical dashed lines). Viruses with lower IC₅₀₋valuesare more susceptible to T-20 than viruses with higher IC₅₀ values.NL4-3: well-characterized X4 tropic strain JRCSF; well-characterized R5tropic strain 91US005.11: R5 tropic isolate obtained from the NIH AIDSResearch and Reference Reagent Program (ARRRP) 92HT593.1: Dual tropic(X4R5) isolate obtained from the NIH ARRRP.92HT599.24: X4 tropic isolateobtained from the NIH ARRRP.

FIG. 4B: Measuring Entry Inhibitor Susceptibility: Drug ResistanceMutations.

In this figure, reduced susceptibility to the fusion inhibitor T-20conferred by specific drug resistance mutations in the gp41 envelopeprotein is demonstrated. Cells expressing CD4, CCR5 and CXCR4 wereinfected in the absence of T-20 and over a wide range of T-20concentrations (x-axis log 10 scale). The percent inhibition of viralreplication (y-axis) was determined by comparing the amount ofluciferase produced in infected cells in the presence of T-20 to theamount of luciferase produced in the absence of T-20. Isogenic virusescontaining one or two specific mutations in the gp41 transmembraneenvelope protein were tested (highlighted in red in the figure legend).Drug susceptibility is quantified by determining the concentration ofT-20 required to inhibit 50% of viral replication (IC₅₀, shown asvertical dashed lines). Viruses with lower IC₅₀ values are moresusceptible to T-20 than viruses with higher IC₅₀ values. No mutation(wildtype sequence): GIV; Single mutations: GIV, DIM, SIV; Doublemutations: DIM, SIM, DTV.

FIG. 5: Fusogenicity Assay.

FIG. 5 presents a diagrammatic representation of a fusogenicity assayperformed to assess the fusogenic activity of HIV envelope proteins.

FIG. 6: Sensitivity or Resistance to Monoclonal Antibody B4, TNX 355,and PRO 542 and Fusogenicity of Sixteen Clones.

FIG. 6 presents a graphical representation of resistance or sensitivityto B4, TNX 355, and PRO542 and fusogenicity of sixteen individual HIVobtained from a single patient sample. The Y-axis for the differentinhibitors represents the IC₅₀ for the drugs, while the fusogenicitypanel represents the fusogenicity of the clones as a percentage offusogenicity observed for reference strain HXB2.

FIG. 7: Alignment of Variable Region 1 (V1) of the Envelope Protein ofSixteen Clones isolated from a Single Patient. Specifically, FIG. 7shows the amino acid sequences of the V1 region, including glycosylationsite motifs contained within, from clones 3, 20, 47, 48, 18, 17, 35, 11,24, and 5 (SEQ ID NO:1), from clone 39 (SEQ ID NO:2), from clones 21 and26 (SEQ ID NO:3), from clone 6 (SEQ ID NO:4), from clone 36 (SEQ IDNO:5), and from clone 43 (SEQ ID NO:6), respectively.

FIG. 7 presents an alignment of variable region 1 from the envelopeprotein of the sixteen clones isolated from a single HIV-infectedsubject. Glycosylation sites are indicated by arrows.

FIG. 8: Alignment of Variable Region 4 (V4) of the Envelope Protein ofSixteen Clones isolated from a Single Patient. Specifically, FIG. 8shows the amino acid sequences of the V4 region, including glycosylationsite motifs contained within, from clones 3, 20, 47, 48, 18, 17, 35, 11and 5 (SEQ ID NO:7); from clone 39 (SEQ ID NO:8); from clone 24 (SEQ IDNO:9). from clones 21, 26, and 6 (SEQ ID NO:10); and from clones 36 and43 (SEQ ID NO:11), respectively.

FIG. 8 presents an alignment of variable region 4 from the envelopeprotein of the sixteen clones isolated from a single HIV-infectedsubject. Glycosylation sites are indicated by the arrows; arrows at thetop of the alignment indicate glycosylation sites present in all clones,while arrows at the bottom of the alignment indicate glycosylation sitespresent in subsets of clones. The left arrow at the bottom indicates aglycosylation site present only in clones 36 and 43; the right arrow atthe bottom indicates a glycosylation site present in all clones shownexcept clone 39.

FIG. 9: Alignment of Variable Region 5 (V5) of the Envelope Protein ofSixteen Clones isolated from a Single Patient. Specifically, FIG. 9shows the amino acid sequences of the V5 region, including glycosylationsite motifs contained within, from clones 3, 20, 39, 47, 48, 18, 17, 35,11, 24, and 5 (SEQ ID NO:12); from clones 21 and 26 (SEQ ID NO:13); fromclone 6 (SEQ ID NO:14); and from clones 36 and 43 (SEQ ID NO:15),respectively.

FIG. 9 presents an alignment of variable region 5 from the envelopeprotein of the sixteen clones isolated from a single HIV-infectedsubject. Glycosylation sites are indicated by the arrows; the arrow atthe top of the alignment indicates a glycosylation site present in allclones, while the arrow at the bottom of the alignment indicates aglycosylation site present in a subset of clones.

FIG. 10: Effects of Changes in Variable Region 5 on Sensitivity toPRO542 and Fusogenicity.

FIG. 10 presents a graphical representation of the effects of changes inthe V5 region of the envelope protein on sensitivity to PRO542 andfusogenicity. Strains A (amino acids 1-14 of SEQ ID NO:13), B (aminoacids 1-10 of SEQ ID NO:12), and C (amino acids 1-14 of SEQ ID NO:15),are individual viral isolates with a V5 sequence as presented in FIG.10. Strains A′ and C′ are strains A and C, respectively, with their V5sequences substituted with the B strain V5 sequence.

FIG. 11: Effects of Changes in Variable Region 5 on Sensitivity toPRO542 and Fusogenicity.

FIG. 11 presents a graphical representation of the effects of changes inthe V5 region of the envelope protein on sensitivity to PRO542 andfusogenicity. Strain B (amino acids 1-10 of SEQ ID NO:12); strain B′(amino acids 1-14 of SEQ ID NO:13) and strain B″ (amino acids 1-14 ofSEQ ID NO:15) are strain B with their V5 sequence replaced with the V5regions of strains A and C, respectively, where strains A and C are alsoas described above in the legend to FIG. 10.

FIG. 12: Effects of Changes in Variable Region 5 on Sensitivity toPRO542 and Fusogenicity.

FIG. 12 presents a graphical representation of the effects of changes inthe V5 region of the envelope protein on sensitivity to PRO542 andfusogenicity. Strain B is as described above in the legend to FIG. 10(amino acids 1-10 of SEQ ID NO:12), strain B′ (amino acids 1-14 of SEQID NO:13) is as described above in the legend to FIG. 11, and strains B³(SEQ ID NO:26) and B⁴ (SEQ ID NO:27) are strain B with their V5sequences replaced with the sequences shown in FIG. 12.

FIG. 13: Suppression of Sensitivity to PRO542 by the L261S Mutation.

FIG. 13 presents log-sigmoid curves showing the sensitivity orresistance to PRO542 in the presence and absence of the L261S mutation.As shown in FIG. 13, an otherwise sensitive virus (IC₅₀ of about 0.9μg/ml) is more resistant to PRO542 (IC₅₀ of about 11 μg/ml) in thepresence of the L261S mutation.

5. DEFINITIONS

As used herein, the following terms shall have the following meanings:

A “phenotypic assay” is a test that measures a phenotype of a particularvirus, such as, for example, HIV, or a population of viruses, such as,for example, the population of HIV infecting a subject. The phenotypesthat can be measured include, but are not limited to, the resistance orsusceptibility of a virus, or of a population of viruses, to a specificanti-viral agent or that measures the replication capacity of a virus.

A “genotypic assay” is an assay that determines a genotype of anorganism, a part of an organism, a population of organisms, a gene, apart of a gene, or a population of genes. Typically, a genotypic assayinvolves determination of the nucleic acid sequence of the relevant geneor genes. Such assays are frequently performed in HIV to establish, forexample, whether certain mutations are associated with drug resistanceor resistance or altered replication capacity are present.

The term “% sequence identity” is used interchangeably herein with theterm “% identity” and refers to the level of amino acid sequenceidentity between two or more peptide sequences or the level ofnucleotide sequence identity between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% identity means the same thing as 80% sequence identitydetermined by a defined algorithm, and means that a given sequence is atleast 80% identical to another length of another sequence. Exemplarylevels of sequence identity include, but are not limited to, 60, 70, 80,85, 90, 95, 98% or more sequence identity to a given sequence.

The term “% sequence homology” is used interchangeably herein with theterm “% homology” and refers to the level of amino acid sequencehomology between two or more peptide sequences or the level ofnucleotide sequence homology between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% homology means the same thing as 80% sequence homologydetermined by a defined algorithm, and accordingly a homologue of agiven sequence has greater than 80% sequence homology over a length ofthe given sequence. Exemplary levels of sequence homology include, butare not limited to, 60, 70, 80, 85, 90, 95, 98% or more sequencehomology to a given sequence.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at the NCBI website. See alsoAltschul et al., 1990, J. Mol. Biol. 215:403-10 (with special referenceto the published default setting, i.e., parameters w=4, t=17) andAltschul et al., 1997, Nucleic Acids Res., 25:3389-3402. Sequencesearches are typically carried out using the BLASTP program whenevaluating a given amino acid sequence relative to amino acid sequencesin the GenBank Protein Sequences and other public databases. The BLASTXprogram is preferred for searching nucleic acid sequences that have beentranslated in all reading frames against amino acid sequences in theGenBank Protein Sequences and other public databases. Both BLASTP andBLASTX are run using default parameters of an open gap penalty of 11.0,and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.See id.

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-X program, operated with default parameters, including anopen gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM30 similarity matrix.

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but which has at least onebond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (O) Ser (S) and Thr (T).

“Nonpolar Amino Acid” refers to a hydrophobic amino acid having a sidechain that is uncharged at physiological pH and which has bonds in whichthe pair of electrons shared in common by two atoms is generally heldequally by each of the two atoms (i.e., the side chain is not polar).Genetically encoded nonpolar amino acids include Ala (A), Gly (G), Ile(I), Leu (L), Met (M) and Val (V).

“Hydrophilic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophilic amino acids include Arg(R), Asn (N), Asp (D), Glu (E), Gln (O), H is (H), Lys (K), Ser (S) andThr (T).

“Hydrophobic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophobic amino acids include Ala(A), Gly (G), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Trp (W), Tyr(Y) and Val (V).

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Asp (D) andGlu (E).

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith a hydrogen ion. Genetically encoded basic amino acids include Arg(R), His (H) and Lys (K).

A “mutation” is a change in an amino acid sequence or in a correspondingnucleic acid sequence relative to a reference nucleic acid orpolypeptide. For embodiments of the invention comprising HIV protease orreverse transcriptase, the reference nucleic acid encoding protease,reverse transcriptase, or envelope is the protease, reversetranscriptase, or envelope coding sequence, respectively, present inNL4-3 HIV (GenBank Accession No. AF324493). Likewise, the referenceprotease, reverse transcriptase, or envelope polypeptide is that encodedby the NL4-3 HIV sequence. Although the amino acid sequence of a peptidecan be determined directly by, for example, Edman degradation or massspectroscopy, more typically, the amino sequence of a peptide isinferred from the nucleotide sequence of a nucleic acid that encodes thepeptide. Any method for determining the sequence of a nucleic acid knownin the art can be used, for example, Maxam-Gilbert sequencing (Maxam etal., 1980, Methods in Enzymology 65:499), dideoxy sequencing (Sanger etal., 1977, Proc. Natl. Acad. Sci. USA 74:5463) or hybridization-basedapproaches (see e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, 3^(rd) ed., NY; andAusubel et al., 1989, Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, NY).

A “mutant” is a virus, gene or protein having a sequence that has one ormore changes relative to a reference virus, gene or protein.

The terms “peptide,” “polypeptide” and “protein” are usedinterchangeably throughout.

The term “wild-type” refers to a viral genotype that does not comprise amutation known to be associated with drug resistance.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout.

As used herein, a “glycosylation site” refers to a single amino acid ora specific sequence of amino acids that is recognized by one skilled inthe art as being suitable for glycosylation as well as a single aminoacid or a specific sequence of amino acids that is actuallyglycosylated.

6. DETAILED DESCRIPTION OF THE INVENTION

In certain aspects, the invention provides a method for determiningwhether an HIV is resistant to an HIV entry inhibitor. The methods areuseful, for example, to guide therapeutic decisions in treatmentsubjects infected with HIV, whether newly infected or failing treatment,for screening compounds to identify compounds that will affect virusesresistant to other entry inhibitors, and to test whether anti-HIVantibodies can neutralize infection by a broad range of HIV that may beresistant to other strategies for treating and/or preventing HIVinfection. Other uses of such methods will be apparent to those of skillin the art.

6.1 Methods for Determining Whether an HIV or HIV Population isResistant to Entry Inhibitors

In one aspect, the invention provides a method for determining whetheran HIV is resistant to an HIV entry inhibitor. In certain aspects, themethod for determining whether an HIV is likely to be more resistant toa CD4 binding site entry inhibitor than a reference HIV comprisescomparing the length of one or more variable regions of an envelopeprotein of the HIV and/or a number of glycosylation sites on theenvelope protein of the HIV to the length of one or more correspondingvariable regions of an envelope protein of the reference HIV or thenumber of glycosylation sites on the envelope protein of the referenceHIV, respectively, wherein the HIV is likely to be more resistant to theCD4 binding site entry inhibitor than the reference HIV when the HIV hasa longer variable region or regions than the reference HIV and/or theHIV has more glycosylation sites than the reference HIV. In certainembodiments, the CD4 binding site entry inhibitor is selected from thegroup consisting of PRO542, TNX-355 and monoclonal antibody B12.Generally, a CD4 binding site inhibitor, as described herein, is anentry inhibitor that competes with CD4 for binding to gp120.Accordingly, in certain embodiments, the CD4 binding site inhibitor canbe any entry inhibitor that competes with CD4 for binding to gp120without limitation. For example, any soluble form of CD4 is a CD4binding site inhibitor. In certain embodiments, the reference HIV isNL4-3, HXB2, or SF2. In certain embodiments, the HIV has longer variableregions than the reference HIV. In certain embodiments, the HIV has moreglycosylation sites than the reference HIV. In certain embodiments, theHIV has longer variable regions and more glycosylation sites than thereference HIV.

In certain embodiments, at least one of the variable regions of the HIVis at least 2 amino acids longer than a corresponding variable region ofthe reference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 5 amino acids longer than a correspondingvariable region of the reference HIV. In certain embodiments, at leastone of the variable regions of the HIV is at least 8 amino acids longerthan a corresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least10 amino acids longer than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 12 amino acids longer than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least15 amino acids longer than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 17 amino acids longer than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least20 amino acids longer than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 22 amino acids longer than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least25 amino acids longer than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 28 amino acids longer than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least30 amino acids longer than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 35 amino acids longer than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least40 amino acids longer than a corresponding variable region of thereference HIV.

In certain embodiments, at least one of the variable regions of the HIVis at least 5% longer than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 10% longer than a corresponding variableregion of the reference HIV. In certain embodiments, at least one of thevariable regions of the HIV is at least 15% longer than a correspondingvariable region of the reference HIV. In certain embodiments, at leastone of the variable regions of the HIV is at least 20% longer than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least25% longer than a corresponding variable region of the reference HIV. Incertain embodiments, at least one of the variable regions of the HIV isat least 30% longer than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 35% longer than a corresponding variableregion of the reference HIV. In certain embodiments, at least one of thevariable regions of the HIV is at least 40% longer than a correspondingvariable region of the reference HIV. In certain embodiments, at leastone of the variable regions of the HIV is at least 45% longer than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least50% longer than a corresponding variable region of the reference HIV. Incertain embodiments, at least one of the variable regions of the HIV isat least 55% longer than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 60% longer than a corresponding variableregion of the reference HIV. In certain embodiments, at least one of thevariable regions of the HIV is at least 65% longer than a correspondingvariable region of the reference HIV. In certain embodiments, at leastone of the variable regions of the HIV is at least 70% longer than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least75% longer than a corresponding variable region of the reference HIV. Incertain embodiments, at least one of the variable regions of the HIV isat least 80% longer than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 85% longer than a corresponding variableregion of the reference HIV. In certain embodiments, at least one of thevariable regions of the HIV is at least 90% longer than a correspondingvariable region of the reference HIV. In certain embodiments, at leastone of the variable regions of the HIV is at least 95% longer than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least100% longer than a corresponding variable region of the reference HIV.In certain embodiments, at least one of the variable regions of the HIVis at least 125% longer than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 150% longer than a corresponding variableregion of the reference HIV. In certain embodiments, at least one of thevariable regions of the HIV is at least 175% longer than a correspondingvariable region of the reference HIV. In certain embodiments, at leastone of the variable regions of the HIV is at least 200% longer than acorresponding variable region of the reference HIV.

In certain embodiments, at least one of the variable regions of the HIVis longer than a corresponding variable region of the reference HIV. Incertain embodiments, at least two of the variable regions of the HIV arelonger than a corresponding variable region of the reference HIV. Incertain embodiments, at least three of the variable regions of the HIVare longer than a corresponding variable region of the reference HIV. Incertain embodiments, at least four of the variable regions of the HIVare longer than a corresponding variable region of the reference HIV. Incertain embodiments, at least five of the variable regions of the HIVare longer than a corresponding variable region of the reference HIV. Incertain embodiments, all of the variable regions of the HIV are longerthan a corresponding variable region of the reference HIV. In certainembodiments, the V1 region of the HIV is longer than the V1 region ofthe reference HIV. In certain embodiments, the V2 region of the HIV islonger than the V2 region of the reference HIV. In certain embodiments,the V3 region of the HIV is longer than the V3 region of the referenceHIV. In certain embodiments, the V4 region of the HIV is longer than theV4 region of the reference HIV. In certain embodiments, the V5 region ofthe HIV is longer than the V5 region of the reference HIV.

In certain embodiments, the HIV's envelope protein comprises at leastone more glycosylation site than the reference HIV's envelope protein.As is well-known in the art, HIV envelope protein is glycosylated at Tor S residues present in the motif N—X-T/S—X, where X is any amino acidthat is not proline. In certain embodiments, the HIV's envelope proteincomprises at least two more glycosylation sites than the reference HIV'senvelope protein. In certain embodiments, the HIV's envelope proteincomprises at least three more glycosylation sites than the referenceHIV's envelope protein. In certain embodiments, the HIV's envelopeprotein comprises at least four more glycosylation sites than thereference HIV's envelope protein. In certain embodiments, the HIV'senvelope protein comprises at least five more glycosylation sites thanthe reference HIV's envelope protein. In certain embodiments, the HIV'senvelope protein comprises at least six more glycosylation sites thanthe reference HIV's envelope protein. In certain embodiments, the HIV'senvelope protein comprises at least seven more glycosylation sites thanthe reference HIV's envelope protein. In certain embodiments, the HIV'senvelope protein comprises at least eight more glycosylation sites thanthe reference HIV's envelope protein. In certain embodiments, the HIV'senvelope protein comprises at least nine more glycosylation sites thanthe reference HIV's envelope protein. In certain embodiments, the HIV'senvelope protein comprises at least ten more glycosylation sites thanthe reference HIV's envelope protein. In certain embodiments, the HIV'senvelope protein comprises at least eleven more glycosylation sites thanthe reference HIV's envelope protein. In certain embodiments, the HIV'senvelope protein comprises at least twelve more glycosylation sites thanthe reference HIV's envelope protein. In certain embodiments, the HIV'senvelope protein comprises at least thirteen more glycosylation sitesthan the reference HIV's envelope protein. In certain embodiments, theHIV's envelope protein comprises at least fourteen more glycosylationsites than the reference HIV's envelope protein. In certain embodiments,the HIV's envelope protein comprises at least fifteen more glycosylationsites than the reference HIV's envelope protein.

In another aspect, the invention provides a method for determiningwhether an HIV is likely to be more resistant to a CD4-blocking entryinhibitor than a reference HIV, comprising comparing the length of oneor more variable regions of an envelope protein of the HIV and/or thenumber of glycosylation sites on the envelope protein of the HIV to thelength of one or more corresponding variable regions of an envelopeprotein of the reference HIV and/or the number of glycosylation sites onthe envelope protein of the reference HIV, respectively, wherein the HIVis likely to be more resistant to the CD4 binding site entry inhibitorthan the reference HIV when the HIV has a shorter variable region orregions than the reference HIV and/or the HIV has fewer glycosylationsites than the reference HIV. In certain embodiments, the entryinhibitor is monoclonal antibody B4. As used herein, a CD4 blockinginhibitor is an entry inhibitor that binds CD4 in a manner that does notcompete with gp120 but nonetheless interferes with CD4-gp120interactions. Accordingly, in certain embodiments, the CD4-blockingentry inhibitor can be any entry inhibitor that binds CD4 in a mannerthat does not compete with gp120 but nonetheless interferes withCD4-gp120 interactions without limitation. In certain embodiments, thereference HIV is NL4-3, HXB2, or SF2. In certain embodiments, the HIVhas shorter variable regions than the reference HIV. In certainembodiments, the HIV has fewer glycosylation sites than the referenceHIV. In certain embodiments, the HIV has shorter variable regions andfewer glycosylation sites than the reference HIV.

In certain embodiments, at least one of the variable regions of the HIVis at least 2 amino acids shorter than a corresponding variable regionof the reference HIV. In certain embodiments, at least one of thevariable regions of the HIV is at least 5 amino acids shorter than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least8 amino acids shorter than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 10 amino acids shorter than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least12 amino acids shorter than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 15 amino acids shorter than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least17 amino acids shorter than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 20 amino acids shorter than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least22 amino acids shorter than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 25 amino acids shorter than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least28 amino acids shorter than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 30 amino acids shorter than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least35 amino acids shorter than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 40 amino acids shorter than acorresponding variable region of the reference HIV.

In certain embodiments, at least one of the variable regions of the HIVis at least 5% shorter than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 10% shorter than a corresponding variableregion of the reference HIV. In certain embodiments, at least one of thevariable regions of the HIV is at least 15% shorter than a correspondingvariable region of the reference HIV. In certain embodiments, at leastone of the variable regions of the HIV is at least 20% shorter than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least25% shorter than a corresponding variable region of the reference HIV.In certain embodiments, at least one of the variable regions of the HIVis at least 30% shorter than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 35% shorter than a corresponding variableregion of the reference HIV. In certain embodiments, at least one of thevariable regions of the HIV is at least 40% shorter than a correspondingvariable region of the reference HIV. In certain embodiments, at leastone of the variable regions of the HIV is at least 45% shorter than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least50% shorter than a corresponding variable region of the reference HIV.In certain embodiments, at least one of the variable regions of the HIVis at least 55% shorter than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 60% shorter than a corresponding variableregion of the reference HIV. In certain embodiments, at least one of thevariable regions of the HIV is at least 65% shorter than a correspondingvariable region of the reference HIV. In certain embodiments, at leastone of the variable regions of the HIV is at least 70% shorter than acorresponding variable region of the reference HIV. In certainembodiments, at least one of the variable regions of the HIV is at least75% shorter than a corresponding variable region of the reference HIV.In certain embodiments, at least one of the variable regions of the HIVis at least 80% shorter than a corresponding variable region of thereference HIV. In certain embodiments, at least one of the variableregions of the HIV is at least 85% shorter than a corresponding variableregion of the reference HIV. In certain embodiments, at least one of thevariable regions of the HIV is at least 90% shorter than a correspondingvariable region of the reference HIV. In certain embodiments, at leastone of the variable regions of the HIV is at least 95% shorter than acorresponding variable region of the reference HIV.

In certain embodiments, at least one of the variable regions of the HIVis shorter than a corresponding variable region of the reference HIV. Incertain embodiments, at least two of the variable regions of the HIV areshorter than a corresponding variable region of the reference HIV. Incertain embodiments, at least three of the variable regions of the HIVare shorter than a corresponding variable region of the reference HIV.In certain embodiments, at least four of the variable regions of the HIVare shorter than a corresponding variable region of the reference HIV.In certain embodiments, at least five of the variable regions of the HIVare shorter than a corresponding variable region of the reference HIV.In certain embodiments, all of the variable regions of the HIV areshorter than a corresponding variable region of the reference HIV. Incertain embodiments, the V1 region of the HIV is shorter than the V1region of the reference HIV. In certain embodiments, the V2 region ofthe HIV is shorter than the V2 region of the reference HIV. In certainembodiments, the V3 region of the HIV is shorter than the V3 region ofthe reference HIV. In certain embodiments, the V4 region of the HIV isshorter than the V4 region of the reference HIV. In certain embodiments,the V5 region of the HIV is shorter than the V5 region of the referenceHIV.

In certain embodiments, the HIV's envelope protein comprises at leastone fewer glycosylation site than the reference HIV's envelope protein.In certain embodiments, the HIV's envelope protein comprises at leasttwo fewer glycosylation sites than the reference HIV's envelope protein.In certain embodiments, the HIV's envelope protein comprises at leastthree fewer glycosylation sites than the reference HIV's envelopeprotein. In certain embodiments, the HIV's envelope protein comprises atleast four fewer glycosylation sites than the reference HIV's envelopeprotein. In certain embodiments, the HIV's envelope protein comprises atleast five fewer glycosylation sites than the reference HIV's envelopeprotein. In certain embodiments, the HIV's envelope protein comprises atleast six fewer glycosylation sites than the reference HIV's envelopeprotein. In certain embodiments, the HIV's envelope protein comprises atleast seven fewer glycosylation sites than the reference HIV's envelopeprotein. In certain embodiments, the HIV's envelope protein comprises atleast eight fewer glycosylation sites than the reference HIV's envelopeprotein. In certain embodiments, the HIV's envelope protein comprises atleast nine fewer glycosylation sites than the reference HIV's envelopeprotein. In certain embodiments, the HIV's envelope protein comprises atleast ten fewer glycosylation sites than the reference HIV's envelopeprotein. In certain embodiments, the HIV's envelope protein comprises atleast eleven fewer glycosylation sites than the reference HIV's envelopeprotein. In certain embodiments, the HIV's envelope protein comprises atleast twelve fewer glycosylation sites than the reference HIV's envelopeprotein. In certain embodiments, the HIV's envelope protein comprises atleast thirteen fewer glycosylation sites than the reference HIV'senvelope protein. In certain embodiments, the HIV's envelope proteincomprises at least fourteen fewer glycosylation sites than the referenceHIV's envelope protein. In certain embodiments, the HIV's envelopeprotein comprises at least fifteen fewer glycosylation sites than thereference HIV's envelope protein.

In certain embodiments, the HIV entry inhibitor binds to a cell surfacereceptor, e.g., CD4, CXCR4, or CCR5. In certain embodiments, thecompound is a ligand of the cell surface receptor. In certainembodiments, the compound comprises an antibody. In certain embodiments,the compound inhibits membrane fusion. In certain embodiments, thecompound is a peptide, a peptidomimetic, an organic molecule, or asynthetic compound. In certain embodiments, the compound binds the viralenvelope protein, e.g., gp120, gp41, and/or gp160.

The invention provides a method for determining whether a virus hasdeveloped resistance to an entry inhibitor which comprises: (a)determining whether a virus is resistant to an entry inhibitor accordingto a method of the invention, wherein a nucleic acid encoding a viralenvelope protein is obtained from a subject at a first time; (b)determining whether a virus is resistant to an entry inhibitor accordinga method of the invention, wherein the nucleic acid encoding the viralenvelope protein is obtained from the subject at a later second time;and (c) comparing the susceptibilities determined in steps (a) and (b),wherein a decrease in susceptibility at the later second time indicatesthat the virus has developed resistance to the entry inhibitor. In aparticular embodiment, the subject has undergone or is undergoinganti-HIV therapy comprising an entry inhibitor. In certain embodiments,the entry inhibitor is a CD4 binding site entry inhibitor. In certainembodiments, the entry inhibitor is a CD4 blocking entry inhibitor.

In another aspect, the invention provides a method for determiningwhether an HIV is likely to exhibit altered susceptibility to an entryinhibitor, comprising detecting, in a nucleic acid encoding an envelopeprotein of the HIV, a mutation in a codon corresponding to codon 261 ofreference HIV strain HXB2, wherein the presence of a mutation in codon261 indicates that the HIV is likely to be resistant to the entryinhibitor.

In certain embodiments, the mutation in codon 261 encodes serine (S). Incertain embodiments, the HIV is an HIV-1. In certain embodiments, theHIV exhibits reduced susceptibility to the entry inhibitor relative to areference HIV. In certain embodiments, the nucleic acid does not encodea mutation at a codon corresponding to codon 639 of reference HIV strainHXB2. In certain embodiments, the nucleic acid does not encode amutation at a codon corresponding to codon 749 of reference HIV strainHXB2. In certain embodiments, the nucleic acid does not encode amutation at a codon corresponding to codon 639 or at codon 749 ofreference HIV strain HXB2. In certain embodiments, the nucleic acid doesnot encode an alanine (A) at a codon corresponding to codon 639 ofreference HIV strain HXB2. In certain embodiments, the nucleic acid doesnot encode an alanine (A) at a codon corresponding to codon 749 ofreference HIV strain HXB2. In certain embodiments, the nucleic acid doesnot encode an alanine (A) at a codon corresponding to codon 639 or atcodon 749 of reference HIV strain HXB2.

In another aspect, the invention provides a method for determiningwhether an HIV is likely to exhibit altered susceptibility to an entryinhibitor, comprising detecting, in a nucleic acid encoding an envelopeprotein of the HIV, a mutation in one or more codons corresponding tocodon 117 and/or at codon 421 of reference HIV strain HXB2, wherein thepresence of the mutations indicates that the HIV is likely to beresistant to the entry inhibitor.

In certain embodiments, the HIV is HIV-1. In certain embodiments, themutation in codon 117 encodes glutamate (E). In certain embodiments, themutation in codon 421 encodes glutamate (E). In certain embodiments, theHIV exhibits increased susceptibility to an entry inhibitor relative toa reference HIV.

In another aspect, the invention provides a method for determiningwhether an HIV is likely to exhibit altered susceptibility to an entryinhibitor, comprising detecting, in a nucleic acid encoding an envelopeprotein of the HIV, a mutation in a codon corresponding to codon 121and/or codon 298 reference HIV strain HXB2, wherein the presence of themutations indicates that the HIV is likely to be resistant to the entryinhibitor.

In certain embodiments, the HIV is HIV-1. In certain embodiments, themutation in codon 121 encodes glutamate (E). In certain embodiments, themutation in codon 298 encodes serine (S). In certain embodiments, theHIV exhibits reduced susceptibility to an entry inhibitor relative to areference HIV.

The invention provides for a method for identifying a mutation in avirus that confers resistance to a compound that inhibits viral entryinto a cell which comprises: (a) determining the nucleic acid sequenceor the amino acid sequence of the virus prior to any treatment of thevirus with the compound; (b) obtaining a virus resistant to thecompound; (c) determining the nucleic acid sequence or the amino acidsequence of the resistant virus from step (b); and (d) comparing thenucleic acid sequence or the amino acid sequences of steps (a) and (c),respectively, so as to identify the mutation in the virus that confersresistance to the compound.

In certain embodiments, the virus obtained in step (b) is the virus ofstep (a) grown in the presence of the compound until resistance isdeveloped.

In certain embodiments, the virus obtained in step (b) is isolated froma subject which has been undergoing treatment with the compound.

In certain embodiments, this invention further provides a means andmethod for discovering, optimizing and characterizing novel or new drugsthat target various defined and as yet undefined steps in the virusattachment and entry process.

In certain embodiments, this invention further provides a means andmethod for discovering, optimizing and characterizing HIV-1 vaccines(either preventative or therapeutic) that target various defined and asyet undefined steps in the virus attachment and entry process.

In certain embodiments, this invention provides a means and method foridentifying amino acid substitutions/mutations in HIV-1 envelopeproteins (gp41 and/or gp120) that alter susceptibility to inhibitors ofvirus entry.

In certain embodiments, this invention further provides a means andmethod for determining HIV-1 envelope amino acid substitutions/mutationsthat are frequently observed, either alone or in combination, in virusesthat exhibit altered susceptibility to virus entry inhibitors.

In certain embodiments, this invention further provides a means andmethod for using virus entry inhibitor susceptibility to guide thetreatment of subjects failing antiretroviral drug treatment.

In certain embodiments, this invention further provides the means andmethods for using virus entry inhibitor susceptibility to guide thetreatment of subjects newly infected with HIV-1.

In another aspect, the methods comprise determining that a subject isinfected with an HIV that is resistant to an HIV entry inhibitoraccording to a method of the invention, then advising a medicalprofessional of the treatment option of administering to the subject atherapeutic regimen that does not include the HIV entry inhibitor. Incertain embodiments, the HIV entry inhibitor is PRO542, TNX-355, mAbB12, or mAb B4. In certain embodiments, the entry inhibitor is selectedfrom the group consisting of BMS-488,403, PRO542, mAb B4, mAb B12,TNX-355, UK-427,857, SCH-D, GW-873,140, AMD-11070, TAK-220, Pro-140, andmAb004. In certain embodiments, the entry inhibitor is selected from thegroup consisting of TNX-355, UK-427,857, SCH-D, GW-873,140, AMD-11070,and TAK-220. In certain embodiments, the entry inhibitor is selectedfrom the group consisting of TNX-355, UK-427,857, SCH-D, GW-873,140, andTAK-220. In certain embodiments, the entry inhibitor is BMS-488,403. Incertain embodiments, the entry inhibitor is PRO542. In certainembodiments, the entry inhibitor is mAb B4. In certain embodiments, theentry inhibitor is TNX-355. In certain embodiments, the entry inhibitoris UK-427,857. In certain embodiments, the entry inhibitor is SCH-D. Incertain embodiments, the entry inhibitor is GW-873,140. In certainembodiments, the entry inhibitor is AMD-11070. In certain embodiments,the entry inhibitor is TAK-220. In certain embodiments, the entryinhibitor is PRO140. In certain embodiments, the entry inhibitor ismAb004. In certain embodiments, the entry inhibitor is mAb B12.

In another aspect, the methods comprise determining that a subject isinfected with an HIV that is resistant to an HIV entry inhibitoraccording to a method of the invention, then advising a medicalprofessional not to treat the subject with the HIV entry inhibitor. Incertain embodiments, the HIV entry inhibitor is PRO542, TNX-355, mAbB12, or mAb B4. In certain embodiments, the entry inhibitor is selectedfrom the group consisting of BMS-488,403, PRO542, mAb B4, mAb B12,TNX-355, UK-427,857, SCH-D, GW-873,140, AMD-11070, TAK-220, Pro-140, andmAb004. In certain embodiments, the entry inhibitor is selected from thegroup consisting of TNX-355, UK-427,857, SCH-D, GW-873,140, AMD-11070,and TAK-220. In certain embodiments, the entry inhibitor is selectedfrom the group consisting of TNX-355, UK-427,857, SCH-D, GW-873,140, andTAK-220. In certain embodiments, the entry inhibitor is BMS-488,403. Incertain embodiments, the entry inhibitor is PRO542. In certainembodiments, the entry inhibitor is mAb B4. In certain embodiments, theentry inhibitor is TNX-355. In certain embodiments, the entry inhibitoris UK-427,857. In certain embodiments, the entry inhibitor is SCH-D. Incertain embodiments, the entry inhibitor is GW-873,140. In certainembodiments, the entry inhibitor is AMD-11070. In certain embodiments,the entry inhibitor is TAK-220. In certain embodiments, the entryinhibitor is PRO140. In certain embodiments, the entry inhibitor ismAb004. In certain embodiments, the entry inhibitor is mAb B12.

In still another aspect, the methods comprise determining that a subjectis infected with an HIV that is resistant to an HIV entry inhibitoraccording to a method of the invention, and administering to the subjecta combination of anti-HIV agents that does not comprise the HIV entryinhibitor. In certain embodiments, the HIV entry inhibitor is PRO542,TNX-355, mAb B12, or mAb B4. In certain embodiments, the entry inhibitoris selected from the group consisting of BMS-488,403, PRO542, mAb B4,mAb B12, TNX-355, UK-427,857, SCH-D, GW-873,140, AMD-11070, TAK-220,Pro-140, and mAb004. In certain embodiments, the entry inhibitor isselected from the group consisting of TNX-355, UK-427,857, SCH-D,GW-873,140, AMD-11070, and TAK-220. In certain embodiments, the entryinhibitor is selected from the group consisting of TNX-355, UK-427,857,SCH-D, GW-873,140, and TAK-220. In certain embodiments, the entryinhibitor is BMS-488,403. In certain embodiments, the entry inhibitor isPRO542. In certain embodiments, the entry inhibitor is mAb B4. Incertain embodiments, the entry inhibitor is TNX-355. In certainembodiments, the entry inhibitor is UK-427,857. In certain embodiments,the entry inhibitor is SCH-D. In certain embodiments, the entryinhibitor is GW-873,140. In certain embodiments, the entry inhibitor isAMD-11070. In certain embodiments, the entry inhibitor is TAK-220. Incertain embodiments, the entry inhibitor is PRO140. In certainembodiments, the entry inhibitor is mAb004. In certain embodiments, theentry inhibitor is mAb B12.

In still another aspect, the methods comprise determining that a subjectis infected with an HIV that is likely to be more resistant to an HIVentry inhibitor than a reference HIV according to a method of theinvention, then advising a medical professional of the treatment optionof administering to the subject a combination of anti-HIV agents thatdoes not comprise an effective amount of the HIV entry inhibitor. Incertain embodiments, the HIV entry inhibitor is PRO542, TNX-355, mAbB12, or mAb B4. In certain embodiments, the entry inhibitor is selectedfrom the group consisting of BMS-488,403, PRO542, mAb B4, mAb B12,TNX-355, UK-427,857, SCH-D, GW-873,140, AMD-11070, TAK-220, Pro-140, andmAb004. In certain embodiments, the entry inhibitor is selected from thegroup consisting of TNX-355, UK-427,857, SCH-D, GW-873,140, AMD-11070,and TAK-220. In certain embodiments, the entry inhibitor is selectedfrom the group consisting of TNX-355, UK-427,857, SCH-D, GW-873,140, andTAK-220. In certain embodiments, the entry inhibitor is BMS-488,403. Incertain embodiments, the entry inhibitor is PRO542. In certainembodiments, the entry inhibitor is mAb B4. In certain embodiments, theentry inhibitor is TNX-355. In certain embodiments, the entry inhibitoris UK-427,857. In certain embodiments, the entry inhibitor is SCH-D. Incertain embodiments, the entry inhibitor is GW-873,140. In certainembodiments, the entry inhibitor is AMD-11070. In certain embodiments,the entry inhibitor is TAK-220. In certain embodiments, the entryinhibitor is PRO140. In certain embodiments, the entry inhibitor ismAb004. In certain embodiments, the entry inhibitor is mAb B12.

In another aspect, the methods comprise determining that a subject isinfected with an HIV that is likely to be more resistant to an HIV entryinhibitor than a reference HIV according to a method of the invention,then advising a medical professional of the treatment option ofadministering to the subject a therapeutic regimen that does not includethe HIV entry inhibitor. In certain embodiments, the HIV entry inhibitoris PRO542, TNX-355, mAb B12, or mAb B4. In certain embodiments, theentry inhibitor is selected from the group consisting of BMS-488,403,PRO542, mAb B4, mAb B12, TNX-355, UK-427,857, SCH-D, GW-873,140,AMD-11070, TAK-220, Pro-140, and mAb004. In certain embodiments, theentry inhibitor is selected from the group consisting of TNX-355,UK-427,857, SCH-D, GW-873,140, AMD-11070, and TAK-220. In certainembodiments, the entry inhibitor is selected from the group consistingof TNX-355, UK-427,857, SCH-D, GW-873,140, and TAK-220. In certainembodiments, the entry inhibitor is BMS-488,403. In certain embodiments,the entry inhibitor is PRO542. In certain embodiments, the entryinhibitor is mAb B4. In certain embodiments, the entry inhibitor isTNX-355. In certain embodiments, the entry inhibitor is UK-427,857. Incertain embodiments, the entry inhibitor is SCH-D. In certainembodiments, the entry inhibitor is GW-873,140. In certain embodiments,the entry inhibitor is AMD-11070. In certain embodiments, the entryinhibitor is TAK-220. In certain embodiments, the entry inhibitor isPRO140. In certain embodiments, the entry inhibitor is mAb004. Incertain embodiments, the entry inhibitor is mAb B12.

In another aspect, the methods comprise determining that a subject isinfected with an HIV that is likely to be more susceptible to an HIVentry inhibitor than a reference HIV according to a method of theinvention, then advising a medical professional to treat the subjectwith the HIV entry inhibitor. In certain embodiments, the HIV entryinhibitor is PRO 542, TNX-355, mAb B12, or mAb B4. In certainembodiments, the entry inhibitor is selected from the group consistingof BMS-488,403, PRO542, mAb B4, mAb B12, TNX-355, UK-427,857, SCH-D,GW-873,140, AMD-11070, TAK-220, Pro-140, and mAb004. In certainembodiments, the entry inhibitor is selected from the group consistingof TNX-355, UK-427,857, SCH-D, GW-873,140, AMD-11070, and TAK-220. Incertain embodiments, the entry inhibitor is selected from the groupconsisting of TNX-355, UK-427,857, SCH-D, GW-873,140, and TAK-220. Incertain embodiments, the entry inhibitor is BMS-488,403. In certainembodiments, the entry inhibitor is PRO542. In certain embodiments, theentry inhibitor is mAb B4. In certain embodiments, the entry inhibitoris TNX-355. In certain embodiments, the entry inhibitor is UK-427,857.In certain embodiments, the entry inhibitor is SCH-D. In certainembodiments, the entry inhibitor is GW-873,140. In certain embodiments,the entry inhibitor is AMD-11070. In certain embodiments, the entryinhibitor is TAK-220. In certain embodiments, the entry inhibitor isPRO140. In certain embodiments, the entry inhibitor is mAb004. Incertain embodiments, the entry inhibitor is mAb B12.

In still another aspect, the methods comprise determining that a subjectis infected with an HIV that is likely to be more susceptible to an HIVentry inhibitor than a reference HIV according to a method of theinvention, and administering to the subject a combination of anti-HIVagents that comprises the HIV entry inhibitor. In certain embodiments,the HIV entry inhibitor is PRO542, TNX-355, mAb B12, or mAb B4. Incertain embodiments, the entry inhibitor is selected from the groupconsisting of BMS-488,403, PRO542, mAb B4, mAb B12, TNX-355, UK-427,857,SCH-D, GW-873,140, AMD-11070, TAK-220, Pro-140, and mAb004. In certainembodiments, the entry inhibitor is selected from the group consistingof TNX-355, UK-427,857, SCH-D, GW-873,140, AMD-11070, and TAK-220. Incertain embodiments, the entry inhibitor is selected from the groupconsisting of TNX-355, UK-427,857, SCH-D, GW-873,140, and TAK-220. Incertain embodiments, the entry inhibitor is BMS-488,403. In certainembodiments, the entry inhibitor is PRO542. In certain embodiments, theentry inhibitor is mAb B4. In certain embodiments, the entry inhibitoris TNX-355. In certain embodiments, the entry inhibitor is UK-427,857.In certain embodiments, the entry inhibitor is SCH-D. In certainembodiments, the entry inhibitor is GW-873,140. In certain embodiments,the entry inhibitor is AMD-11070. In certain embodiments, the entryinhibitor is TAK-220. In certain embodiments, the entry inhibitor isPRO140. In certain embodiments, the entry inhibitor is mAb004. Incertain embodiments, the entry inhibitor is mAb B12.

In still another aspect, the methods comprise determining that a subjectis infected with an HIV that is likely to be more susceptible to an HIVentry inhibitor than a reference HIV according to a method of theinvention, then advising a medical professional of the treatment optionof administering to the subject a combination of anti-HIV agents thatcomprises an effective amount of the HIV entry inhibitor. In certainembodiments, the HIV entry inhibitor is PRO542, TNX-355, mAb B12, or mAbB4. In certain embodiments, the entry inhibitor is selected from thegroup consisting of BMS-488,403, PRO542, mAb B4, mAb B12, TNX-355,UK-427,857, SCH-D, GW-873,140, AMD-11070, TAK-220, Pro-140, and mAb004.In certain embodiments, the entry inhibitor is selected from the groupconsisting of TNX-355, UK-427,857, SCH-D, GW-873,140, AMD-11070, andTAK-220. In certain embodiments, the entry inhibitor is selected fromthe group consisting of TNX-355, UK-427,857, SCH-D, GW-873,140, andTAK-220. In certain embodiments, the entry inhibitor is BMS-488,403. Incertain embodiments, the entry inhibitor is PRO542. In certainembodiments, the entry inhibitor is mAb B4. In certain embodiments, theentry inhibitor is TNX-355. In certain embodiments, the entry inhibitoris UK-427,857. In certain embodiments, the entry inhibitor is SCH-D. Incertain embodiments, the entry inhibitor is GW-873,140. In certainembodiments, the entry inhibitor is AMD-11070. In certain embodiments,the entry inhibitor is TAK-220. In certain embodiments, the entryinhibitor is PRO 140. In certain embodiments, the entry inhibitor ismAb004. In certain embodiments, the entry inhibitor is mAb B12.

In yet another aspect, the methods comprise determining that a subjectis infected with an HIV that is likely to be more susceptible to an HIVentry inhibitor than a reference HIV according to a method of theinvention, then advising a medical professional of the treatment optionof administering to the subject an effective amount of the HIV entryinhibitor. In certain embodiments, the HIV entry inhibitor is PRO542,TNX-355, mAb B12, or mAb B4. In certain embodiments, the entry inhibitoris selected from the group consisting of BMS-488,403, PRO542, mAb B4,mAb B12, TNX-355, UK-427,857, SCH-D, GW-873,140, AMD-11070, TAK-220,Pro-140, and mAb004. In certain embodiments, the entry inhibitor isselected from the group consisting of TNX-355, UK-427,857, SCH-D,GW-873,140, AMD-11070, and TAK-220. In certain embodiments, the entryinhibitor is selected from the group consisting of TNX-355, UK-427,857,SCH-D, GW-873,140, and TAK-220. In certain embodiments, the entryinhibitor is BMS-488,403. In certain embodiments, the entry inhibitor isPRO542. In certain embodiments, the entry inhibitor is mAb B4. Incertain embodiments, the entry inhibitor is TNX-355. In certainembodiments, the entry inhibitor is UK-427,857. In certain embodiments,the entry inhibitor is SCH-D. In certain embodiments, the entryinhibitor is GW-873,140. In certain embodiments, the entry inhibitor isAMD-11070. In certain embodiments, the entry inhibitor is TAK-220. Incertain embodiments, the entry inhibitor is PRO140. In certainembodiments, the entry inhibitor is mAb004. In certain embodiments, theentry inhibitor is mAb B12.

In still another aspect, the methods comprise determining whether asubject is infected with an HIV that is likely to be more resistant toan HIV entry inhibitor than a reference HIV according to a method of theinvention at a first time, then determining whether the subject remainsinfected with an HIV that is likely to be more resistant to an HIV entryinhibitor than a reference HIV according to a method of the invention ata later second time. In other embodiments, the methods comprisedetermining whether a subject is infected with an HIV that is likely tobe less resistant to an HIV entry inhibitor than a reference HIVaccording to a method of the invention at a first time, then determiningwhether the subject is infected with an HIV that is likely to be moreresistant to an HIV entry inhibitor than a reference HIV according to amethod of the invention at a later second time. In certain embodiments,the entry inhibitor is a CD4 binding site inhibitor as described herein.In certain embodiments, the entry inhibitor is a CD4 blocking inhibitoras described herein.

In yet another aspect, the methods comprise determining whether asubject is infected with an HIV that is likely to be more susceptible toan HIV entry inhibitor than a reference HIV according to a method of theinvention at a first time, then determining whether the subject remainsinfected with an HIV that is likely to be more susceptible to an HIVentry inhibitor than a reference HIV according to a method of theinvention at a later second time. In other embodiments, the methodscomprise determining whether a subject is infected with an HIV that islikely to be more susceptible to an HIV entry inhibitor than a referenceHIV according to a method of the invention at a first time, thendetermining whether the subject is infected with an HIV that is likelyto be more susceptible to an HIV entry inhibitor than a reference HIVaccording to a method of the invention at a later second time. Incertain embodiments, the entry inhibitor is a CD4 binding site inhibitoras described herein. In certain embodiments, the entry inhibitor is aCD4 blocking inhibitor as described herein. In a certain embodiments,the subject has undergone or is undergoing anti-HIV therapy comprisingan entry inhibitor. In certain embodiments, the entry inhibitor is a CD4binding site entry inhibitor. In certain embodiments, the entryinhibitor is a CD4 blocking entry inhibitor.

In still another aspect, the invention provides a method for identifyingcompounds that can inhibit HIV entry into a cell expressing a receptorthat is bound by an HIV envelope protein, comprising performing an entryassay (e.g., as described in Example 1) with an HIV that is more likelyto be resistant to an entry inhibitor than a reference HIV as determinedwith a method of the invention. In still another aspect, the inventionprovides a method for identifying compounds that can inhibit HIV entryinto a cell expressing a receptor that is bound by an HIV envelopeprotein, comprising performing an entry assay (e.g., as described inExample 1) with an HIV that is less likely to be susceptible to an entryinhibitor than a reference HIV as determined with a method of theinvention. In certain embodiments, the entry inhibitor is a CD4 bindingsite inhibitor as described herein. In certain embodiments, the entryinhibitor is a CD4 blocking inhibitor as described herein.

In yet another aspect, the invention provides a method of assessing anantibody response for its ability to neutralize infection. Methods forperforming such assays are extensively described in U.S. applicationSer. Nos. 10/077,027 and 10/504,821. In certain embodiments, the assaysare performed with an HIV that is more likely to be resistant to anentry inhibitor than a reference HIV as determined with a method of theinvention. In certain embodiments, the assays are performed with an HIVthat is more likely to be susceptible to an entry inhibitor than areference HIV as determined with a method of the invention. In certainembodiments, the entry inhibitor is a CD4 binding site inhibitor asdescribed herein. In certain embodiments, the entry inhibitor is a CD4blocking inhibitor as described herein.

6.2 Determining Viral Variable Region or Glycosylation Genotypes

The length of envelope protein variable regions and/or number ofenvelope protein glycosylation sites and/or the sequence of envelopeprotein variable or constant regions can be determined by any meansknown in the art for detecting a mutation. For example, the length ofenvelope protein variable regions and/or number of envelope proteinglycosylation sites can be detected in the viral gene that encodes aparticular protein, or in the protein itself, i.e., in the amino acidsequence of the protein.

A length of envelope protein variable regions and/or number of envelopeprotein glycosylation sites within the env gene can be detected byutilizing any suitable technique known to one of skill in the artwithout limitation. Viral DNA or RNA can be used as the starting pointfor such assay techniques, and may be isolated according to standardprocedures which are well known to those of skill in the art.

The determination of specific nucleic acid sequences, such as in aparticular region of the env gene, can be accomplished be a variety ofmethods including, but not limited to,restriction-fragment-length-polymorphism detection based onallele-specific restriction-endonuclease cleavage (Kan and Dozy, 1978,Lancet ii:910-912), mismatch-repair detection (Faham and Cox, 1995,Genome Res 5:474-482), binding of MutS protein (Wagner et al., 1995,Nucl Acids Res 23:3944-3948), denaturing-gradient gel electrophoresis(Fisher et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1579-83),single-strand-conformation-polymorphism detection (Orita et al., 1983,Genomics 5:874-879), RNAase cleavage at mismatched base-pairs (Myers etal, 1985, Science 230:1242), chemical (Cotton et al., 1988, Proc. Natl.Acad. Sci. U.S.A. 85:4397-4401) or enzymatic (Youil et al., 1995, Proc.Natl. Acad. Sci. U.S.A. 92:87-91) cleavage of heteroduplex DNA, methodsbased on oligonucleotide-specific primer extension (Syvanen et al.,1990, Genomics 8:684-692), genetic bit analysis (Nikiforov et al., 1994,Nucl Acids Res 22:4167-4175), oligonucleotide-ligation assay (Landegrenet al., 1988, Science 241:1077), oligonucleotide-specific ligation chainreaction (“LCR”) (Barrany, 1991, Proc. Natl. Acad. Sci. U.S.A.88:189-193), gap-LCR (Abravaya et al., 1995, Nucl Acids Res 23:675-682),radioactive or fluorescent DNA sequencing using standard procedures wellknown in the art, and peptide nucleic acid (PNA) assays (Orum et al.,1993, Nucl. Acids Res. 21:5332-5356; Thiede et al., 1996, Nucl. AcidsRes. 24:983-984).

In addition, viral DNA or RNA may be used in hybridization oramplification assays to detect abnormalities involving gene structure,including point mutations, insertions, deletions and genomicrearrangements. Such assays may include, but are not limited to,Southern analyses (Southern, 1975, J. Mol. Biol. 98:503-517), singlestranded conformational polymorphism analyses (SSCP) (Orita et al.,1989, Proc. Natl. Acad. Sci. USA 86:2766-2770), and PCR analyses (U.S.Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188; PCRStrategies, 1995 Innis et al. (eds.), Academic Press, Inc.).

Such diagnostic methods can involve for example, contacting andincubating the viral nucleic acids with one or more labeled nucleic acidreagents including recombinant DNA molecules, cloned genes or degeneratevariants thereof, under conditions favorable for the specific annealingof these reagents to their complementary sequences. Preferably, thelengths of these nucleic acid reagents are at least 15 to 30nucleotides. After incubation, all non-annealed nucleic acids areremoved from the nucleic acid molecule hybrid. The presence of nucleicacids which have hybridized, if any such molecules exist, is thendetected. Using such a detection scheme, the nucleic acid from the viruscan be immobilized, for example, to a solid support such as a membrane,or a plastic surface such as that on a microtiter plate or polystyrenebeads. In this case, after incubation, non-annealed, labeled nucleicacid reagents of the type described above are easily removed. Detectionof the remaining, annealed, labeled nucleic acid reagents isaccomplished using standard techniques well-known to those in the art.The gene sequences to which the nucleic acid reagents have annealed canbe compared to the annealing pattern expected from a normal genesequence in order to determine the length of envelope protein variableregions and/or number of envelope protein glycosylation sites in the envgene.

These techniques can easily be adapted to provide high-throughputmethods for determining the length of envelope protein variable regionsand/or number of envelope protein glycosylation sites in viral genomes.For example, a gene array from Affymetrix (Affymetrix, Inc., Sunnyvale,Calif.) can be used to rapidly identify genotypes of a large number ofindividual viruses. Affymetrix gene arrays, and methods of making andusing such arrays, are described in, for example, U.S. Pat. Nos.6,551,784, 6,548,257, 6,505,125, 6,489,114, 6,451,536, 6,410,229,6,391,550, 6,379,895, 6,355,432, 6,342,355, 6,333,155, 6,308,170,6,291,183, 6,287,850, 6,261,776, 6,225,625, 6,197,506, 6,168,948,6,156,501, 6,141,096, 6,040,138, 6,022,963, 5,919,523, 5,837,832,5,744,305, 5,834,758, and 5,631,734, each of which is herebyincorporated by reference in its entirety.

In addition, Ausubel et al., eds., Current Protocols in MolecularBiology, 2002, Vol. 4, Unit 25B, Ch. 22, which is hereby incorporated byreference in its entirety, provides further guidance on construction anduse of a gene array for determining the genotypes of a large number ofviral isolates. Finally, U.S. Pat. Nos. 6,670,124; 6,617,112; 6,309,823;6,284,465; and 5,723,320, each of which is incorporated by reference inits entirety, describe related array technologies that can readily beadapted for rapid identification of a large number of viral genotypes byone of skill in the art.

Alternative diagnostic methods for the detection of gene specificnucleic acid molecules may involve their amplification, e.g., by PCR(U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188; PCRStrategies, 1995 Innis et al. (eds.), Academic Press, Inc.), followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art. The resulting amplified sequences can becompared to those which would be expected if the nucleic acid beingamplified contained only normal copies of the respective gene in orderto determine the length of envelope protein variable regions and/ornumber of envelope protein glycosylation sites.

Additionally, the nucleic acid can be sequenced by any sequencing methodknown in the art. For example, the viral DNA can be sequenced by thedideoxy method of Sanger et al., 1977, Proc. Natl. Acad. Sci. USA74:5463, as further described by Messing et al., 1981, Nuc. Acids Res.9:309, or by the method of Maxam et al., 1980, Methods in Enzymology65:499. See also the techniques described in Sambrook et al., 2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,3^(rd) ed., NY; and Ausubel et al., 1989, Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley Interscience, NY.

Antibodies directed against the viral gene products, i.e., viralproteins or viral peptide fragments can also be used to detect thelength of envelope protein variable regions and/or number of envelopeprotein glycosylation sites in the viral proteins. Alternatively, theviral protein or peptide fragments of interest can be sequenced by anysequencing method known in the art in order to yield the amino acidsequence of the protein of interest. An example of such a method is theEdman degradation method which can be used to sequence small proteins orpolypeptides. Larger proteins can be initially cleaved by chemical orenzymatic reagents known in the art, for example, cyanogen bromide,hydroxylamine, trypsin or chymotrypsin, and then sequenced by the Edmandegradation method.

Further, glycosylation of particular viral peptides can be detected byany method known to one of skill in the art without limitation. Forexample, conventional mass spectroscopy or NMR techniques can be used todetect the presence and/or identify of glycosylated viral peptides orproteins. See, e.g., Rusnak et al., 2002, J. Biomol. Tech.13(4):228-237.

6.3 Computer-Implemented Methods for Determining Whether a Virus isResistant to an Entry Inhibitor and Articles Related Thereto

In another aspect, the present invention provides computer-implementedmethods for determining whether an HIV is resistant to an entryinhibitor. In such embodiments, the methods of the invention are adaptedto take advantage of the processing power of modern computers. One ofskill in the art can readily adapt the methods in such a manner.Therefore, in certain embodiments, the invention provides acomputer-implemented method for determining whether an HIV is likely tobe more resistant to a CD4 binding site entry inhibitor than a referenceHIV, comprising inputting genotypic information into a memory system ofa computer, wherein the genotypic information comprises the length of atleast one variable region of the envelope protein of the HIV and/or thenumber of glycosylation sites of the envelope protein of the HIV and thelength of the corresponding at least one variable region of the envelopeprotein of the reference HIV and/or the number of glycosylation sites ofthe envelope protein of the reference HIV, respectively; comparing thelength of the at least one variable region of the envelope proteins ofthe HIV or the number of glycosylation sites of the envelope protein ofthe HIV to the length of the corresponding at least one variable regionof the envelope protein of the HIV and/or the number of glycosylationsites of the envelope protein of the reference HIV, and determiningwhether the HIV is resistant to the entry inhibitor, wherein the HIV islikely to be more resistant to the CD4 binding site inhibitor than thereference HIV if the at least one variable region of the HIV is longerthan the corresponding at least one variable region of the reference HIVand/or the HIV's envelope protein comprises more glycosylation sitesthan the reference HIV's envelope protein.

In other embodiments, the invention provides a computer-implementedmethod for determining whether an HIV is likely to be more resistant toa CD4-blocking entry inhibitor than a reference HIV, comprisinginputting genotypic information into a memory system of a computer,wherein the genotypic information comprises the length of at least onevariable region of the envelope protein of the HIV and/or the number ofglycosylation sites of the envelope protein of the HIV and the length ofa corresponding at least one variable region of the envelope protein ofthe reference HIV and/or the number of glycosylation sites of theenvelope protein of the reference HIV, respectively; comparing thelength of the at least one variable region of the envelope proteins ofthe HIV or the number of glycosylation sites of the envelope protein ofthe HIV to the length of the corresponding at least one variable regionof the envelope protein of the HIV and/or the number of glycosylationsites of the envelope protein of the reference HIV, and determiningwhether the HIV is resistant to the entry inhibitor, wherein the HIV islikely to be more resistant to the CD4-blocking entry inhibitor than thereference HIV if the at least one variable region of the HIV is shorterthan the corresponding at least one variable region of the reference HIVand/or the HIV's envelope protein comprises fewer glycosylation sitesthan the reference HIV's envelope protein.

In certain embodiments, the methods further comprise displaying whetherthe HIV is likely to be more resistant to an HIV entry inhibitor than areference HIV on a display of the computer. In certain embodiments, themethods further comprise printing whether the HIV is likely to be moreresistant to an HIV entry inhibitor than a reference HIV.

In another aspect, the invention provides a tangible medium indicatingwhether an HIV is likely to be more resistant to an HIV entry inhibitorthan a reference HIV produced according to a method of the invention. Incertain embodiments, the tangible medium is a computer-readable medium.In certain embodiments, the tangible medium is a paper document. Incertain embodiments, the paper document is a printed document, e.g., acomputer print-out. In still another aspect, the invention provides acomputer-readable medium comprising data indicating whether an HIV islikely to be more resistant to an HIV entry inhibitor than a referenceHIV produced according to a method of the invention.

In yet another aspect, the invention provides a computer-readable mediumthat comprises data indicating whether an HIV is likely to be moreresistant to an HIV entry inhibitor than a reference HIV producedaccording a method of the invention. In certain embodiments, thecomputer-readable medium is a random-access memory. In certainembodiments, the computer-readable medium is a fixed disk. In certainembodiments, the computer-readable medium is a floppy disk. In certainembodiments, the computer-readable medium is a portable memory device,such as, e.g., a USB key or an iPod™.

In still another aspect, the invention provides an article ofmanufacture that comprises computer-readable instructions for performinga method of the invention. In certain embodiments, the article ofmanufacture is a random-access memory. In certain embodiments, thearticle of manufacture is a fixed disk. In certain embodiments, thearticle of manufacture is a floppy disk. In certain embodiments, thearticle of manufacture is a portable memory device, such as, e.g., a USBkey or an iPod™.

In yet another aspect, the invention provides a computer-readable mediumthat comprises data indicating whether an HIV is likely to be moreresistant to an HIV entry inhibitor than a reference HIV andcomputer-readable instructions for performing a method of the invention.In certain embodiments, the computer-readable medium is a random-accessmemory. In certain embodiments, the computer-readable medium is a fixeddisk. In certain embodiments, the computer-readable medium is a floppydisk. In certain embodiments, the computer-readable medium is a portablememory device, such as, e.g., a USB key or an iPod™.

In yet another aspect, the invention provides a computer system that isconfigured to perform a method of the invention.

6.4 Viruses and Viral Samples

The length of envelope protein variable regions and/or number ofenvelope protein glycosylation sites can be determined from a viralsample obtained by any means known in the art for obtaining viralsamples. Such methods include, but are not limited to, obtaining a viralsample from a human or an animal infected with the virus or obtaining aviral sample from a viral culture. In one embodiment, the viral sampleis obtained from a human individual infected with the virus. The viralsample could be obtained from any part of the infected individual's bodyor any secretion expected to contain the virus. Examples of such partsinclude, but are not limited to blood, serum, plasma, sputum, lymphaticfluid, semen, vaginal mucus and samples of other bodily fluids. In apreferred embodiment, the sample is a blood, serum or plasma sample.

In another embodiment, the length of envelope protein variable regionsand/or number of envelope protein glycosylation sites is determined froma virus that can be obtained from a culture. In some embodiments, theculture can be obtained from a laboratory. In other embodiments, theculture can be obtained from a collection, for example, the AmericanType Culture Collection. In certain embodiments, the length of envelopeprotein variable regions and/or number of envelope protein glycosylationsites is determined for NL4-3, SF2, or HXB2.

In certain embodiments, the length of envelope protein variable regionsand/or number of envelope protein glycosylation sites is determined froma derivative of a virus. In one embodiment, the derivative of the virusis not itself pathogenic. In another embodiment, the derivative of thevirus is a plasmid-based system, wherein replication of the plasmid orof a cell transfected with the plasmid is affected by the presence orabsence of the selective pressure, such that mutations are selected thatincrease resistance to the selective pressure. In some embodiments, thederivative of the virus comprises the nucleic acids or proteins ofinterest, for example, those nucleic acids or proteins to be targeted byan anti-viral treatment. In one embodiment, the genes of interest can beincorporated into a vector. See, e.g., U.S. application Ser. Nos.09/874,475 and 10/077,027, each of which is incorporated herein byreference. In certain embodiments, the genes can be those that encodeenvelope protein (gp160).

In another embodiment, the intact virus need not be used. Instead, apart of the virus incorporated into a vector can be used. Preferablythat part of the virus is used that is targeted by an anti-viral drug.

In another embodiment, the length of envelope protein variable regionsand/or number of envelope protein glycosylation sites is determined in agenetically modified virus. The virus can be genetically modified usingany method known in the art for genetically modifying a virus. Forexample, the virus can be grown for a desired number of generations in alaboratory culture. In one embodiment, no selective pressure is applied(i.e., the virus is not subjected to a treatment that favors thereplication of viruses with certain characteristics), and new mutationsaccumulate through random genetic drift. In another embodiment, aselective pressure is applied to the virus as it is grown in culture(i.e., the virus is grown under conditions that favor the replication ofviruses having one or more characteristics). In one embodiment, theselective pressure is an anti-viral treatment. Any known anti-viraltreatment can be used as the selective pressure.

In certain embodiments, the virus is HIV and the selective pressure is aNNRTI. In another embodiment, the virus is HIV-1 and the selectivepressure is a NNRTI. Any NNRTI can be used to apply the selectivepressure. Examples of NNRTIs include, but are not limited to,nevirapine, delavirdine and efavirenz. By treating HIV cultured in vitrowith a NNRTI, one can select for mutant strains of HIV that have anincreased resistance to the NNRTI. The stringency of the selectivepressure can be manipulated to increase or decrease the survival ofviruses not having the selected—for characteristic.

In other embodiments, the virus is HIV and the selective pressure is aNRTI. In another embodiment, the virus is HIV-1 and the selectivepressure is a NRTI. Any NRTI can be used to apply the selectivepressure. Examples of NRTIs include, but are not limited to, AZT, ddI,ddC, d4T, 3TC, abacavir, and tenofovir. By treating HIV cultured invitro with a NRTI, one can select for mutant strains of HIV that have anincreased resistance to the NRTI. The stringency of the selectivepressure can be manipulated to increase or decrease the survival ofviruses not having the selected-for characteristic.

In still other embodiments, the virus is HIV and the selective pressureis a PI. In another embodiment, the virus is HIV-1 and the selectivepressure is a PI. Any PI can be used to apply the selective pressure.Examples of P is include, but are not limited to, saquinavir, ritonavir,indinavir, nelfinavir, amprenavir, lopinavir and atazanavir. By treatingHIV cultured in vitro with a PI, one can select for mutant strains ofHIV that have an increased resistance to the PI. The stringency of theselective pressure can be manipulated to increase or decrease thesurvival of viruses not having the selected-for characteristic.

In still other embodiments, the virus is HIV and the selective pressureis an entry inhibitor. In another embodiment, the virus is HIV-1 and theselective pressure is an entry inhibitor. Any entry inhibitor can beused to apply the selective pressure. An example of a entry inhibitorincludes, but is not limited to, fusion inhibitors such as, for example,enfuvirtide. Other entry inhibitors include co-receptor inhibitors, suchas, for example, AMD3100 (AnorMED). Such co-receptor inhibitors caninclude any compound that interferes with an interaction between HIV anda co-receptor, e.g., CCR5 or CRCX4, without limitation. Still otherentry inhibitors include UK-427857 (Pfizer), TNX-355 (Tanox Inc.),AMD-070 (AnorMED), Pro 140 (Progenics), FP-21399 (EMD Lexigen),BMS-488043 (Bristol-Myers Squibb), and GSK-873,140 (GlaxoSmithKline). Bytreating HIV cultured in vitro with an entry inhibitor, one can selectfor mutant strains of HIV that have an increased resistance to the entryinhibitor. The stringency of the selective pressure can be manipulatedto increase or decrease the survival of viruses not having theselected-for characteristic.

In another aspect, a mutation associated with NNRTI hypersusceptibilityaccording to the present invention can be made by mutagenizing a virus,a viral genome, or a part of a viral genome. Any method of mutagenesisknown in the art can be used for this purpose. In certain embodiments,the mutagenesis is essentially random. In certain embodiments, theessentially random mutagenesis is performed by exposing the virus, viralgenome or part of the viral genome to a mutagenic treatment. In anotherembodiment, a gene that encodes a viral protein that is the target of ananti-viral therapy is mutagenized. Examples of essentially randommutagenic treatments include, for example, exposure to mutagenicsubstances (e.g., ethidium bromide, ethylmethanesulphonate, ethylnitroso urea (ENU) etc.) radiation (e.g., ultraviolet light), theinsertion and/or removal of transposable elements (e.g., Tn5, Tn10), orreplication in a cell, cell extract, or in vitro replication system thathas an increased rate of mutagenesis. See, e.g., Russell et al., 1979,Proc. Nat. Acad. Sci. USA 76:5918-5922; Russell, W., 1982, EnvironmentalMutagens and Carcinogens: Proceedings of the Third InternationalConference on Environmental Mutagens. One of skill in the art willappreciate that while each of these methods of mutagenesis isessentially random, at a molecular level, each has its own preferredtargets.

In another aspect, the length of envelope protein variable regionsand/or number of envelope protein glycosylation sites can be determinedin an HIV or HIV derivative made using site-directed mutagenesis. Anymethod of site-directed mutagenesis known in the art can be used (seee.g., Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, 3^(rd) ed., NY; and Ausubel et al., 1989,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, NY). See, e.g., Sarkar and Sommer, 1990,Biotechniques, 8:404-407. The site directed mutagenesis can be directedto, e.g., a particular gene or genomic region, a particular part of agene or genomic region, or one or a few particular nucleotides within agene or genomic region. In one embodiment, the site directed mutagenesisis directed to a viral genomic region, gene, gene fragment, ornucleotide based on one or more criteria. In one embodiment, a gene or aportion of a gene is subjected to site-directed mutagenesis because itencodes a protein that is known or suspected to be a target of ananti-viral therapy, e.g., the gene encoding the HIV reversetranscriptase. In another embodiment, a portion of a gene, or one or afew nucleotides within a gene, are selected for site-directedmutagenesis. In one embodiment, the nucleotides to be mutagenized encodeamino acid residues that are known or suspected to interact with ananti-viral compound. In another embodiment, the nucleotides to bemutagenized encode amino acid residues that are known or suspected to bemutated in viral strains that are resistant or susceptible orhypersusceptible to one or more antiviral agents. In another embodiment,the mutagenized nucleotides encode amino acid residues that are adjacentto or near in the primary sequence of the protein residues known orsuspected to interact with an anti-viral compound or known or suspectedto be mutated in viral strains that are resistant or susceptible orhypersusceptible to one or more antiviral agents. In another embodiment,the mutagenized nucleotides encode amino acid residues that are adjacentto or near to in the secondary, tertiary or quaternary structure of theprotein residues known or suspected to interact with an anti-viralcompound or known or suspected to be mutated in viral strains having analtered replication capacity. In another embodiment, the mutagenizednucleotides encode amino acid residues in or near the active site of aprotein that is known or suspected to bind to an anti-viral compound.

7. EXAMPLES

The following examples are presented to further illustrate and explainthe present invention and should not be taken as limiting in any regard.Certain of these experiments were also described in U.S. applicationSer. Nos. 09/874,475 and 10/077,027, each of which is incorporated byreference in its entirety.

7.1 Example 1

Measuring Phenotypic Drug Susceptibility to Inhibitors of HIV-1 Entry

This example provides a means and method for accurately and reproduciblymeasuring susceptibility to inhibitors of HIV-1 attachment and entry(heretofore collectively referred to as entry). Based on this example,the means and method for measuring susceptibility to inhibitors of HIV-1entry can be adapted to other viruses, including, but not limited toother lentiviruses (e.g. HIV-2), other retroviruses (e.g. HTLV-1 and 2),hepadnaviruses (human hepatitis B virus), flaviviruses (human hepatitisC virus) and herpesviruses human cytomegalovirus). This example furtherprovides a means and method for measuring alterations (increases anddecreases) in susceptibility to entry inhibitors.

Measurements of entry inhibitor susceptibility are carried out usingadaptations of the means and methods for phenotypic drug susceptibilityand resistance tests described in U.S. Pat. No. 5,837,464 (InternationalPublication Number WO 97/27319) which is hereby incorporated byreference.

One vector, an example of the envelope expression vector, (pHIVenv) isdesigned to express the envelope polyprotein (gp160) encoded by subjectderived HIV envelope sequences (FIG. 1). Gp160 is subsequently cleavedby a cellular protease to generate the surface (gp120SU) andtransmembrane (gp41™) subunits that comprise the envelope protein on thesurface of HIV-1 virus particles. A second vector, an example of theviral expression vector, (either pHIVluc or pHIVluc.DELTΔA.U3) isdesigned to express genomic and subgenomic viral RNAs and all HIVproteins except the envelope polyprotein (FIGS. 1A-1B).

In this application, patient-derived segment(s) correspond to the codingregion (˜2.5 KB) of the HIV-1 envelope polyprotein (gp160) and representeither (a) envelope sequences amplified by the reversetranscription-polymerase chain reaction method CRT-PCR) using viral RNAisolated from virus derived from HIV-infected individuals, or (b)envelope sequences derived from molecular clones of HIV-1 that containspecific mutations introduced by site directed mutagenesis of a parentalmolecular clone (typically NL4-3).

Isolation of viral RNA was performed using standard procedures (e.g.RNAgents Total RNA Isolation System, Promega, Madison Wis. or RNAzol,Tel-Test, Friendswood, Tex.). The RT-PCR protocol was divided into twosteps. A retroviral reverse transcriptase [e.g. Superscript II(Invitrogen, Life Technologies) Moloney MuLV reverse transcriptase(Roche Molecular Systems, Inc., Branchburg, N.J.), or avianmyeloblastosis virus (AMV) reverse transcriptase, (Boehringer Mannheim,Indianapolis, Ind.)] was used to copy viral RNA into first strand cDNA.The cDNA was then amplified to high copy number using a thermostable DNApolymerase [e.g. Taq (Roche Molecular Systems, Inc., Branchburg, N.J.),Tth (Roche Molecular Systems, Inc., Branchburg, N.J.), PrimeZyme(isolated from Thermus brockianus, Biometra, Gottingen, Germany)] or acombination of thermostable polymerases as described for the performanceof “long PCR” (Barnes, W. M., (1994) Proc. Natl. Acad. Sci, USA 91,2216-2220) [e.g. Expand High Fidelity PCR System (Taq+Pwo), (BoehringerMannheim. Indianapolis, Ind.) OR GeneAmp XL PCR kit (Tth+Vent), (RocheMolecular Systems, Inc., Branchburg, N.J.), Advantage-2, (CloneTech).

Oligo-dT was used for reverse transcription of viral RNA into firststrand cDNA. Envelope PCR primers, forward primer Xho/Pin and reverseprimer Mlu/Xba (Table 3) were used to amplify the patient-derivedsegments. These primers are designed to amplify the ˜2.5 kB envelopegene encoding the gp160 envelope polyprotein, while introducing Xho Iand Pin AI recognition sites at the 5′ end of the PCR amplificationproduct, and Mlu I and Xba I sites at the 3′ end of the PCRamplification product.

Subject derived segments (2.5 kB envelope sequence amplificationproduct) were inserted into HIV-1 envelope expression vectors usingrestriction endonuclease digestion, DNA ligation and bacterialtransformation methods as described in U.S. Pat. No. 5,837,464(International Publication Number WO 97/27319), with minor adaptations.The ˜2.5 kB amplification product was digested with either Xho I or PinAI at the 5′ end and either Mlu I or Xba I at the 3′ end. The resultingdigestion products were ligated, using DNA ligase, into the 5′ Xho I/PinAI and 3′ Mlu I/Xba I sites of modified pCXAS or pCXAT expressionvectors. The construction of the pCXAS and pCXAT vectors has beendescribed in U.S. Pat. No. 5,837,464 (International Publication NumberWO 97/27319)). Modified pCXAS and pCXAT vectors contain a Pin AIrestriction site in addition to the Xho I, Mlul and Xba I restrictionsites that exist in pCXAS and pCXAT. The Pin AI site was introducedbetween the Xho I and Mlu I sites by site directed mutagenesis, suchthat the four sites are located 5′ to 3′ in the following order; Xho I,Pin AI, Mlu I and Xba I. In a preferred embodiment, the 2.5 kBamplification products were digested with Pin AI and Mlu I and ligatedinto the 5′ Pin AI site and the 3′ Mlu I site of the modified pCXASexpression vector. Ligation reaction products were used to transform E.coli. Following a 24-36 h incubation period at 30-37.degree. C., theexpression vector plasmid DNA was purified from the E. coli cultures. Toensure that expression vector preparations adequately represents the HIVquasi-species present in the serum of a given subject, many (>100)independent E. coli transformants were pooled and used for thepreparations of pHIVenv plasmid DNA. Vectors that are assembled in thismanner for the purposes of expressing subject virus derived envelopeproteins are collectively referred to as pHIVenv (FIGS. 1 and 3).

The genomic HIV expression vectors pHIVluc and pHIVlucΔU3 are designedto transcribe HIV genomic RNA and subgenomic mRNAs and to express allHIV proteins except the envelope polyprotein (FIG. 1B). In thesevectors, a portion of the envelope gene has been deleted to accommodatea functional indicator gene cassette, in this case, “Firefly Luciferase”that is used to monitor the ability of the virus to replicate in thepresence or absence of anti-viral drugs. In pHIVlucΔU3, a portion of the3′ U3 region has been deleted to prevent transcription of viral RNAsfrom the 5′ LTR in infected cells.

Susceptibility assays for HIV-1 entry inhibitors were performed usingpackaging host cells consisting of the human embryonic kidney cell line293 (Cell Culture Facility, UC San Francisco, SF, Calif.) and targethost cells consisting of a human osteosarcoma (HOS) cell line expressingCD4 (HT4) plus CCR5, and CXCR4, or astrocytoma (U-87) cell linesexpressing either CD4 and CCR5 or CD4 and CXCR4.

Drug susceptibility testing was performed using pHIVenv and pHIVluc orpHIVlucΔU3. Pseudotyped HIV particles containing envelope proteinsencoded by the subject derived segment were produced by transfecting apackaging host cell (HEK 293) with resistance test vector DNA. Virusparticles were collected (˜48 h) after transfection and are used toinfect target cells (HT4/CCR5/CXCR4, or U-87/CD4/CXCR4, orU-87/CD4/CCR5) that express HIV receptors (i.e. CD4) and co-receptors(i.e. CXCR4, CCR5). After infection (˜72 h) the target cells are lysedand luciferase activity is measured. HIV must complete one round ofreplication to successfully infect the target host cell and produceluciferase activity. The amount of luciferase activity detected in theinfected cells is used as a direct measure of “infectivity” (FIGS. 1 and2). If for any reason (e.g. lack of the appropriate receptor orco-receptor, inhibitory drug activity, neutralizing antibody binding),the virus is unable to enter the target cell, luciferase activity isdiminished. Drug susceptibility is assessed by comparing the infectivityin the absence of drug to infectivity in the presence of drug. Relativedrug susceptibility can be quantified by comparing the susceptibility ofthe “test” virus to the susceptibility of a well-characterized referencevirus (wildtype) derived from a molecular clone of HIV-1, for exampleNL4-3 or HXB2.

Packaging host cells were seeded in 10-cm-diameter dishes and weretransfected one day after plating with pHIVenv and pHIVluc orpHIVlucΔU3. Transfections were performed using a calcium-phosphateco-precipitation procedure. The cell culture media containing the DNAprecipitate was replaced with fresh medium, from one to 24 hours, aftertransfection. Cell culture media containing viral particles wastypically harvested 2 days after transfection and was passed through a0.45-mm filter. Before infection, target cells were plated in cellculture media. Entry inhibitor drugs were typically added to targetcells at the time of infection (one day prior to infection on occasion).Typically, 3 days after infection target cells were assayed forluciferase activity using the Steady-Glo reagent (Promega) and aluminometer.

7.2 Example 2

Identifying Envelope Amino Acid Substitutions/Mutations that AlterSusceptibility to Virus Entry Inhibitors

This example provides a means and method for identifying mutations inHIV-1 envelope that confer reduced susceptibility/resistance to virusentry inhibitors. This example also provides a means and method forquantifying the degree of reduced susceptibility to entry inhibitorsconferred by specific envelope mutations.

Envelope sequences derived from subject samples, or individual clonesderived from subject samples, or envelope sequences engineered by sitedirected mutagenesis to contain specific mutations, were tested in theentry assay to quantify drug susceptibility based on awell-characterized reference standard (e.g. NL4-3, HXB2).

In one embodiment, susceptibility to longitudinal subject samples(viruses collected from the same subject at different timepoints) isevaluated. For example, susceptibility to entry inhibitors is measuredprior to initiating therapy, before or after changes in drug treatment,or before or after changes in virologic (RNA copy number), immunologic(CD4 T-cells), or clinical (opportunistic infection) markers of diseaseprogression.

7.2.1 Genotypic Analysis of Subject HIV Samples

Envelope sequences representing subject sample pools, or clones derivedfrom subject pools, can be analyzed by any broadly available DNAsequencing methods. In this example, subject HIV sample sequences weredetermined using viral RNA purification, RT/PCR and dideoxynucleotidechain terminator sequencing chemistry and capillary gel electrophoresis(Applied Biosystems, Foster City, Calif.). Envelope sequences of subjectvirus pools or clones were compared to reference sequences and othersubject samples. The genotypes of the viruses were examined forsequences that are different from the reference or pre-treatmentsequence and correlated to differences in entry inhibitorsusceptibility, as described below.

7.2.2 Entry Inhibitor Susceptibility of Site Directed Mutants

Genotypic changes that correlate with changes in fitness are evaluatedby constructing envelope expression vectors (pHIVenv) containing thespecific mutation on a defined, drug susceptible, genetic background(e.g. NL4-3 reference strain). Mutations may be incorporated aloneand/or in combination with other mutations that are thought to modulatethe entry inhibitor susceptibility. Envelope mutations are introducedinto pHIVenv vectors using any of the broadly available methods forsite-directed mutagenesis. In certain embodiments the mega-primer PCRmethod for site-directed mutagenesis is used (Sarkar, G. and Summer, S.S., 1990). A pHIVenv vector containing a specific envelope mutation orgroup of mutations are tested using the virus entry assay described inExample 1. Drug susceptibility of the virus containing envelopemutations is compared to the drug susceptibility of a geneticallydefined drug susceptible virus that lacks the specific mutations underevaluation. Observed changes in entry inhibitor susceptibility areattributed to the specific mutations introduced into the pHIVenv vector.

7.3 Example 3

Measuring Susceptibility to Virus Entry Inhibitors to Guide TreatmentDecisions

This example provides a means and method for using virus entry inhibitorsusceptibility to guide the treatment of HIV-1. This example furtherprovides a means and method for using virus entry inhibitorsusceptibility to guide the treatment of subjects that have receivedprevious antiretroviral treatment with a virus entry inhibitor. Thisinvention further provides the means and methods for using virus entryinhibitor susceptibility to guide the treatment of subjects that havenot received previous treatment with a virus entry inhibitor.

In one embodiment, the susceptibility of subject's viruses to virusentry inhibitors is used to guide the treatment of subjects failingantiretroviral regimens that include one or more virus entry inhibitors.Treatment failure (also referred to as virologic failure) is generallydefined as partially suppressive antiviral treatment resulting indetectable levels of virus, which is typically measured in the subjectplasma). Guidance may include, but is not limited to, (a) clarificationof available drug treatment options, (b) selection of more activetreatment regimens, (c) clarification of the etiology of rising viralload in treated subjects (i.e. poor adherence, drug resistance), and (d)reduction in the use of inactive and potentially toxic drugs. In thisembodiment, resistance test vectors are derived from a subject virussamples and tested for susceptibility to various virus entry inhibitorsusing the phenotypic virus entry assay. Virus entry inhibitors mayinclude, but are not limited to, fusion inhibitors (e.g. T-20, T-1249),co-receptors antagonists (AMD3100, AMD8664, TAK779, PRO542, andpeperidin-lyl butane compounds) and CD4 antagonists (MAb B4).Appropriate treatment decisions are based on the results of the virusentry assay (e.g. see FIG. 4B) and additional relevant laboratory testresults and clinical information.

In another embodiment, the susceptibility of subject's viruses to virusentry inhibitors is used to guide the treatment of subjects that havenot been previously treated with antiretroviral regimens that includeone or more virus entry inhibitors. Guidance may include, but is notlimited to, (a) clarification of available drug treatment options, (b)selection of more active treatment regimens, (c) clarification of thebaseline susceptibility to virus entry inhibitors, and (d) reduction inthe use of inactive and potentially toxic drugs. Determining baselinesusceptibility of virus entry inhibitors in treatment naive subjects isimportant for two reasons. First, the natural susceptibility of virusesto entry inhibitors can vary widely (e.g. see FIG. 4A). Second, theincreased use of virus entry inhibitors will undoubtedly result in thegeneration of drug resistant variants that can be transmitted to newlyinfected individuals. In this embodiment, resistance test vectors arederived from a subject virus samples and tested for susceptibility tovarious virus entry inhibitors using the phenotypic virus entry assay.Virus entry inhibitors may include, but are not limited to, fusioninhibitors (e.g. T-20, T-1249), co-receptors antagonist (e.g. AMD3100,AMD8664, TAK-355, PRO542, and peperidin-lyl butane compounds) and CD4antagonists (MAb 5A8). Appropriate treatment decisions are based on theresults of the virus entry assay and additional relevant laboratory testresults and clinical information.

7.4 Example 4

Assays Assessing Fusogenicity of HIV Envelope Proteins

This example provides means and methods for determining thefusogenicity, e.g., the propensity of a virus to mediate aggregation ofsusceptible cells and fusion of their membranes to form syncytia, of avirus's envelope protein. Briefly, nucleic acids encoding HIV-1 envelopeproteins were amplified from patient plasma and introduced to anexpression vector as described above in Example 1. See FIG. 5. Membranefusion was measured by co-culturing HEK-293 cells transientlytransfected with an HIV genomic vector comprising a deletion of the envgene (similar to pHIVluc, described above, but lacking luciferaseactivity) and the expression vector and CD4/CCR5/CXCR4-positive 5.25cells transformed with plasmids expressing both green fluorescentprotein (GFP) and luciferase under control of the HIV-1 long terminalrepeat (LTR). Fusion was visualized by detecting GFP activity andquantified by determining amounts of luciferase activity.

More specifically, the fusion assay was developed by replacing the U87target cell line used in the entry assay with the 5.25.Luc4.M7 cellline. The 5.25.Luc4.M7 cell line was derived from the CEMX174 cell lineand contains green fluorescent protein (GFP) and firefly luciferase(LUC) reporter genes. GFP and LUC expression in 5.25.Luc4.M7 cells isregulated by tat-induction of the HIV-1 LTR. The fusion assay wasperformed by co-transfecting 293 effector cells with an envelope deletedHIV genomic vector plus an HIV envelope expression vector. At 48 hourspost-transfection, the 293 effector cells were harvested, washedthoroughly to remove virus particles, and added to 5.25.Luc4.M7 targetcell cultures in the presence of a reverse transcriptase inhibitor toinhibit virus replication. Membrane fusion was triggered in theco-cultures by the interaction of the HIV envelope protein produced inthe 293 effector cells with the CD4, CCR5 and/or CXCR4 expressed on thesurface of the 5.25.Luc4.M7 target cells. The tat protein derived fromthe effector cells trans-activates the GFP and LUC reporter genes. Inthis assay, cell-cell fusion can be observed directly using fluorescencemicroscopy to view GFP expression, and can be accurately quantifiedusing luciferase (or immuno-chemical detection of GFP).

7.5 Example 5

Identifying Determinants of Fusogenicity or Resistance to EntryInhibitors

This example provides methods and compositions for determining increasedor decreased fusogenicity and/or increased or decreased susceptibilityto viral entry inhibitors. Binding and entry of appropriate cells wasassessed as described in Example 1, while fusogenicity was tested asdescribed in Example 4. To determine genotypes of the envelope proteinsfor which entry and fusogenicity phenotypes were determined, envelopegene sequences were determined using viral RNA purification, RT/PCR andABI chain terminator automated sequencing according to conventionalprotocols as described in Example 2.

To define genetic and structural determinants that confer differentialsensitivity to CD4-gp120 inhibition and membrane fusion, 16 envelopemolecular clones derived from a virus population infecting a singlepatient were sequenced as described above and tested for susceptibilityto entry inhibitors and for fusogenicity. In particular, susceptibilityto CD4 binding site inhibitors (PRO542, a soluble form of CD4; ProgenicsPharmaceuticals, Inc.; Tarrytown, N.Y.; and IgG-b12; Scripps ResearchInstitute; La Jolla, Calif.) and anti-CD4 monoclonal antibody B4 (UnitedBioMedical, Inc.; Hauppauge, N.Y.) was assessed using these assays. Inaddition, the envelope genes of these sixteen clones were sequenced asdescribed above and the sequence of the encoded envelope protein wasdeduced. Phylogenetic analysis revealed two related virus populationsthat segregated based on sensitivity to CD4-gp120 inhibitors. Insummary, clonal and chimeric envelopes containing shorter gp120 variableloops and fewer glycosylation sites exhibited increased susceptibilityto CD4-binding site inhibitors PRO542 and IgG-b12 but decreasedsusceptibility to the anti-CD4 monoclonal antibody B4. Further, theenvelopes containing shorter gp120 variable loops induced higher levelsof membrane fusion.

First, the sensitivity of the 16 different clones to the three differententry inhibitors was assessed using the methods described in Example 1.As shown in FIG. 6, the sixteen clones exhibited increasing resistanceto the anti-CD4 mAb B4 from clone 1 to clone 16. Sensitivity to TNX-355and PRO542 generally varied inversely to sensitivity to B4. Further,fusogenicity generally corresponded to sensitivity to B4 and variedinversely to sensitivity to TNX 355 and PRO542. Two samples, clones 1and 4, exhibited altered susceptibility to PRO542 that did not correlatewell with resistance to TNX 355 and susceptibility to B4; these clonesre believed to have an as-yet uncharacterized mutation that is believedto cause resistance to TNX 355 in an otherwise susceptible geneticbackground. Experiments to identify the genetic determinants of TNX 355resistance in these clones are presently ongoing.

To assess the genetic basis of the differences in susceptibilityobserved in the sixteen clones, the sequences of the envelope genesencoding the envelope proteins of the sixteen clones were determined andcompared. At the outset, it should be noted that the sixteen clones ofFIG. 6 are the same as the clones of FIG. 7-9. The correspondence of theclones is as follows: clone 1 of FIG. 6 is clone 43 of FIGS. 7-9; clone2 of FIG. 6 is clone 24 of FIGS. 7-9; clone 3 of FIG. 6 is clone 11 ofFIGS. 7-9; clone 4 of FIG. 6 is clone 36 of FIGS. 7-9; clone 5 of FIG. 6is clone 26 of FIGS. 7-9; clone 6 of FIG. 6 is clone 6 of FIGS. 7-9;clone 7 of FIG. 6 is clone 21 of FIGS. 7-9; clone 8 of FIG. 6 is clone 3of FIGS. 7-9; clone 9 of FIG. 6 is clone 18 of FIGS. 7-9; clone 10 ofFIG. 6 is clone 17 of FIGS. 7-9; clone 11 of FIG. 6 is clone 35 of FIGS.7-9; clone 12 of FIG. 6 is clone 20 of FIGS. 7-9; clone 13 of FIG. 6 isclone 5 of FIGS. 7-9; clone 14 of FIG. 6 is clone 48 of FIGS. 7-9; clone15 of FIG. 6 is clone 47 of FIGS. 7-9; and clone 16 of FIG. 6 is clone39 of FIGS. 7-9.

FIG. 7 presents an alignment of variable region 1 (V1) of the envelopeproteins of the sixteen samples. As shown in FIG. 7, the 11 clones thatexhibited increased fusogenicity, sensitivity to PRO542, and resistanceto B4 had shorter variable regions than the 5 clones that exhibitedincreased fusogenicity, sensitivity to PRO542, and resistance to B4.

In addition, the differences in length of the V1 region between the twosets of clones altered the distances between glycosylation sites in theregion. As is well-known in the art, HIV envelope protein is heavilyglycosylated at well-defined positions. The motif that is glycosylatedcan be represented with the formula N—X-T/S—X, where N is asparagine, Tis threonine, S is serine, and X is any amino acid that is not proline.The T or S residue in the motif is the point of attachment for N-linkedglycosylation. The glycosylation sites in variable regions of the HIVenvelope protein shown in the figures are indicated with bold type. Asshown in FIG. 7, the length present in the less fusogenic, resistant toPRO542, and sensitive to B4 clones results in separation ofglycosylation sites by an additional 23 amino acids relative to morefusogenic, sensitive to PRO542, and resistant to B4 clones. Thisseparation of the glycosylation sites may additionally contribute to thephenotypes observed for the less fusogenic, resistant to PRO542, andsensitive to B4 clones.

Further, the sequences of variable region 4 (V4) of the sixteen clones'envelope proteins were also aligned as shown in FIG. 8. As shown in FIG.8, clones more fusogenic, sensitive to PRO542, and resistant to B4 hadslightly shorter V4 regions relative to less fusogenic, resistant toPRO542, and sensitive to B4 clones. Further, several of the morefusogenic, sensitive to PRO542, and resistant to B4 clones comprisedmutations that eliminated glycosylation motifs present in the lessfusogenic, resistant to PRO542, and sensitive to B4 clones. For example,the last two clones (identified as clones 36 and 43), which wereresistant to PRO542 and sensitive to B4, comprised two glycosylationsites not present in the first 11 clones (clones 3, 20, 39, 47, 48, 18,17, 35, 11, 24, and 5). In addition, the V4 region of the last twoclones (clones 36 and 43) were three amino acids longer than the V4regions of the first 11 clones, again indicating that the length of thevariable regions of the envelope protein affects fusogenicity,sensitivity to PRO542, and resistance to B4. However, the twelfththrough fourteenth clones (clones 21, 26, and 6) comprised only oneglycosylation motif not present in the first 11 clones and were the samelength as the first 11 clones.

Alignments of variable region 5 (V5) of the sixteen clones' envelopeproteins are presented as FIG. 9. Similar to the observations for the V1and V4 regions, clones more fusogenic, sensitive to PRO542, andresistant to B4 had slightly shorter V5 regions relative to lessfusogenic, resistant to PRO542, and sensitive to B4 clones. Further, theresidues present in the less fusogenic, resistant to PRO542, andsensitive to B4 clones added a glycosylation motif not present in themore fusogenic, sensitive to PRO542, and resistant to B4 clones. Thus,consistent with the observations from the V1 and V4 regions, addedlength to the V5 regions and/or additional glycosylation motifs presentin the V5 region resulted in less fusion, resistance to PRO542, andsensitivity to B4.

To explore the relative contributions of length and glycosylation tothis phenomenon, several recombinant clones were constructed. Ingeneral, the recombinant clones were constructed by swapping the V5region of different clones using conventional techniques. The results ofthese swapping experiments are shown in FIGS. 10-13. FIG. 10 presentsthe effects of a domain swapping experiment on fusogenicity and PRO542sensitivity. Three strains, A, B, and C, served as the basis for theexperiment presented in FIG. 10; strain A is resistant to PRO542 andexhibits low fusogenicity, strain B is sensitive to PRO542 and exhibitshigh fusogenicity, and strain C is moderately sensitive to PRO542 andexhibits low fusogenicity. The portion of the V5 region shown in FIG. 10from strain B was swapped into the remainder of the envelope proteinfrom strains A and C, respectively. As shown in FIG. 10, swapping thisportion of strain B′s V5 region into strain A and C to form strains A′and C′ both shortens the V5 region and destroys a glycosylation site. Asalso shown in FIG. 10, strain A′ exhibits increased sensitivity toPRO542 and increased fusogenicity relative to strain A, demonstratingthat shortening of the V5 region and/or deletion of a glycosylation siteresults in increased sensitivity to PRO542 and increased fusogenicity.Similarly, strain C′ also exhibits increased sensitivity to PRO542 andincreased fusogenicity relative to strain C, confirming this result.

To assess whether the phenotypic differences observed between strain Aand strain C result from the different sequences present in V5 in thesestrains (seen in FIG. 10) or from differences in other envelope regions,two additional constructs, strains B′ and B″ were constructed. Strain B′was constructed to contain the V5 region of strain A in an other wisestrain B background, and strain B″ was constructed to contain the V5region of strain C in an otherwise strain B background. As shown in FIG.11, strains B′ and B″ exhibit essentially identical susceptibility toPRO542 and fusogenicity, demonstrating that differences between strainA′s and strain C′s susceptibility to PRO542 and fusogenicity result fromdifferences in the envelope protein other than in V5. Thus, the sequencevariation observed in V5 between strain A and strain C does not appearto affect susceptibility to PRO542 and fusogenicity; rather, thepresence or absence of the additional length and/or glycosylation sitein V5 was responsible for the differences observed between strains A andC and strain B.

Two additional strains, B¹ and B⁴, Were constructed. As shown in FIG.12, strains B³ and B⁴ were constructed to comprise altered V5 regions:strain B³ includes four amino acids in V5 that are not present in strainB but do encode not a glycosylation site, while strain B includes fouramino acids not present in strain B and also comprises an extraglycosylation site. As shown in FIG. 11, strains B³ and B⁴ each exhibitsubstantially reduced fusogenicity relative to strain B, while strain B⁴exhibits reduced sensitivity to PRO542. Interestingly, strain B′exhibits further reductions in fusogenicity and sensitivity to PRO542,indicating that such phenotypes can also be influenced by the sequencepresent in the V5 region. Nonetheless, FIG. 11 demonstrates thatadditional length and/or additional glycosylation sites in V5 resultedin reduced fusogenicity and increased resistance to PRO 542.

Finally, two clones presented in FIG. 6 exhibited divergent results fromthe remainder of the clones and were therefore subjected to furtheranalysis. In particular, clones 2 and 3 (clones 24 and 11 in thealignments of FIG. 7-9) were predicted to be sensitive to PRO542 basedon their relative lack of glycosylation and short variable loops.However, both clones 2 and 3 were resistant to PRO542 (see FIG. 6).Genotypic analysis revealed that both clones 1 and 4 contained a singlemutation in constant region 2 (C2) of the envelope protein (L261S) thatwas not present in other clones with V1, V4, and V5 similar to those ofclones 1 and 4. Position 261 in this clone corresponds to amino acid 262the envelope protein of a reference HIV strain, NL4-3 (Accession No.AAB60578).

To assess the role of the L261S mutation in suppressing thePRO542-sensitive phenotype of viruses with short variable regions and/orfew glycosylation sites, a recombinant envelope gene was constructedthat comprised the wild-type residue at position 261 of C2 in the samegenetic background as clone 3 of FIG. 6 (Clone 11 of FIGS. 7-9).Reversion of the L261S mutation to wild-type restored sensitivity toPRO542 of clone 3, as shown in FIG. 13. Thus, clones 2 and 3 both hadshort variable regions and few glycosylation sites in those regions,like, for example, clone 1, but L261S in clones 2 and 3 suppressed thePRO542-sensitive phenotype predicted from the short variable regions andfew glycosylation sites.

7.6 Example 6

Characterization of Determinants for Fusogenicity and Resistance toEntry Inhibitors

This example describes the results of experiments designed to identifyand characterize particular molecular determinants for fusogenicity,infectivity, and resistance to entry inhibitors. In the experiments,individual patient-derived envelope genes prepared according to Example1 were characterized as described in Examples 2, 3, and 4. To assess therelative contributions of mutations present in gp120 and/or gp41 toaltered fusogenicity, infectivity, and susceptibility to entryinhibitors, chimeric envelope genes were constructed to encode a portionof the gp120 from one envelope gene isolate while holding the remainderof the gene constant. To ensure that no unrecognized mutations wereintroduced through this procedure, the nucleotide sequences of therecombinant clones were verified as described in Example 2. The resultsof these experiments are presented in Tables 4-Y, below. In the Tables,infectivity results are presented as a raw number of relativefluorescence units observed, fusogenicity is presented as a percentageof fusogenicity observed relative to reference strain HXB2, andsusceptibility to a representative entry inhibitor, PRO542, is presentedas the IC₅₀.

In the experiments presented in Table 4, the interaction between theL261S mutation and mutations at positions 639 and 749 were tested.First, the envelope gene sequences of two clones (clone 4 and clone 5)from a single patient were determined as described above. The envelopeproteins encoded by these genes had identical sequences except forvariance at positions 261, 639, and 749, numbered as the residues arefound in the gp160 polyprotein. The numbering of these residuescorresponds to the numbering found in reference strain HXB2 (AccessionNo. AAB50262).

TABLE 4 Phenotypes gp120 gp41 PRO 542 Clone ID 261 mutations 639 749mutations RFU Fusion, % (IC50 ug/ml) Clone 4 S L261S T V 135,982 1 53.7Clone 5 L A A T639A, V749A 542,286 18 1.09 Clone 6 L T V 680,088 34 0.42Clone 7 S L261S A A T639A, V749A 237,648 7 0.40 HXB2 L T V

The residues at these positions, together with data showing infectivity(RFU), fusogenicity, and IC₅₀ for PRO542 shown in Table 4. Also shown inTable 4 is the amino acid found at these positions in reference strainHXB2. To confirm the effects of the L261S mutation on infectivity,fusogenicity, and susceptibility to PRO542, the portion of the envelopegene from clone 5 comprising the 261 position was introduced into clone4 to form clone 6. As shown in Table 4, reversion of L261S to L resultedin increased infectivity, fusogenicity, and susceptibility to PRO542relative to clone 4.

To test the interactions between mutations at position 261 and positions639 and 749, the portion of the envelope gene comprising the L261Smutation from clone 4 was introduced into clone 5 to form clone 7. Asshown in Table 4, combination of the L261S mutation with T639A and V749Aresulted in increased infectivity, fusogenicity, and susceptibility toPRO 542 relative to clone 4, containing the L261S mutation alone. Thus,the combination of T639A and V749A appears to suppress the reducedinfectivity, fusogenicity, and susceptibility to PRO542 phenotypesobserved for the L261S mutation.

In another set of experiments, the interactions between variants atpositions 117, 421, 741, and 854 were assessed. The residues present atthese positions and the infectivity, fusogenicity, and susceptibility toPRO542 phenotypes are presented in Table 5.

TABLE 5 Phenotypes gp120 gp41 PRO 542 Clone ID 117 421 mutations 741 854mutations RFU Fusion, % (IC50 ug/ml) clone 8 E E K117E, K421E D P I854P9,855 2 0.0062 clone 9 K K G L D741G, I854L 599,179 46 0.27 clone 10 K KD L I854L 680,088 34 0.42 clone 11 E K K117E D L I854L 17,931 1 4.00clone 12 E E K117E, K421E D L I854L 8,377 2 0.0055 clone 13 K E K421E DL I854L 1,310 1 DNR HXB2 K K D I

Clones 8 and 9 were each single clones isolated from a patient. Theclones were identical except for the variance at positions 117, 421,741, and 854 as shown in Table 5, above. Clone 8 comprised threemutations, K117E, K421E, and 1854P, and exhibited reduced infectivityand fusogenicity and increased susceptibility to PRO542 relative toClone 9. Clone 9 comprised two mutations relative to HXB2, D741G and1854L.

To assess the relative contributions of the variant amino acids, clones10, 11, 12, and 13 were constructed by cloning appropriate portions ofclone 8 into a clone 9 background, or vice versa. First, the effects ofthe D741G mutation were assessed by comparing clone 10 to clone 9. Asshown in Table 5, this mutation does not appear to significantly affectany of the tested phenotypes in the tested genetic background.

Next, the effects of the K117E and K421E mutations were tested bycomparing clones 11, 12, and 13 to clone 10. As shown in Table 5, theK117E mutation alone (clone 11) resulted in reduced infectivity,fusogenicity, and PRO542 susceptibility relative to clone 10. The K421Emutation alone (clone 13) resulted in reduced infectivity andfusogenicity relative to clone 10; the IC₅₀ for PRO542 could not bedetermined for this clone because of its very low infectivity. Thecombination of K117E and K421E (clone 12) resulted in reducedinfectivity and fusogenicity but increased PRO542 susceptibilityrelative to clone 10.

In another set of experiments, the relative contributions of variance atpositions 121 and 298 were assessed for the same phenotypes as describedabove. The results of these experiments are presented in Table 6.

TABLE 6 Phenotypes gp120 Fusion, PRO 542 Clone ID 121 298 mutations RFU% (IC50 ug/ml) clone 14 K G R298G 6,938 19 0.0082 clone 15 E R K121E96,981 1 70.0 clone 16 K R 144,580 2 33.5 clone 17 E G K121E, 71,151 30.023 R298G clone 18 K G R298G 113,619 18 0.0055 clone 19 E G K121E,7,687 14 0.010 R298G clone 20 K R 192,028 3 16.0 HXB2 K R

Clones 14 and 15 were each single envelope clones isolated from the samepatient. To begin to assess the relative contributions of the mutationsobserved in clones 14 and 15 (R298G and K121E, respectively), a seriesof chimeric envelope genes were constructed and their phenotypesdetermined as described above. As shown in Table 6, clone 14 exhibitsreduced infectivity, increased fusogenicity, and increasedsusceptibility to PRO542 relative to clone 15.

First, clone 16 was constructed by replacing the region of clone 15comprising position 121 with the corresponding region from clone 14.Reversion of the K121E mutation to wild-type in the clone 15 backgroundresulted in increased infectivity, fusogenicity, and susceptibility toPRO542 relative to clone 15. Next, clone 17 was constructed by replacingthe region of clone 15 comprising position 298 with the correspondingregion from clone 14. The combination of the K121E mutation and theR298G mutation resulted in decreased infectivity, increasedfusogenicity, and greatly increased susceptibility to PRO542 relative toclone 15. To confirm that the change in fusogenicity and susceptibilityto PRO542 resulted from the K121E, mutation and not some otherdifference in envelope, clone 18 was constructed by cloning the regionof clone 14 comprising positions 121 and 298 into clone 15. As expected,clone 18 showed increased fusogenicity and susceptibility to PRO542relative to clone 15. Interestingly, clone 18 exhibited substantiallyincreased infectivity relative to clone 14.

Next, clone 19 was constructed by replacing the region of clone 14comprising position 298 with the corresponding region from clone 15.Clone 19 exhibited relatively similar infectivity, fusogenicity, andsusceptibility to clone 14. Finally, clone 20 was constructed by wasconstructed by replacing the region of clone 14 comprising position 121with the corresponding region from clone 15. Clone 20 exhibitedincreased infectivity and reduced fusogenicity and susceptibility toPRO542 relative to clone 14. Thus, reversion of the K121E mutation inthe genetic background of clone 14 resulted in increased infectivity andreduced fusogenicity and susceptibility to PRO542.

Next the effects of pairs of mutations at positions 164 and 165 and atpositions 344 and 346 were compared in a series of experimentssummarized in Table 7, below.

TABLE 7 Phenotypes gp120 PRO 542 Clone ID 164, 165 344, 346 mutationsRFU Fusion, % (IC50 ug/ml) Clone 21 II RA S164I, Q344R 25,409 74 0.034Clone 22 VV QV S164V, I165V, 146,086 25 4.64 A346V Clone 23 VV RA S164V,I165V, 261,832 51 0.68 Q344R Clone 24 II QV A164V, I165V, 107,502 532.03 A346V Clone 25 II QV S164I, A346V 76,813 46 0.050 Clone 26 II RAS164I, Q344R 78,126 90 0.038 HXB2 SI QA

Clones 21 and 22 were each single envelope clones isolated from the samepatient. To begin to assess the relative contributions of the mutationspresent in clones 21 and 22 to the infectivity, fusogenicity, andsusceptibility to PRO542, clones 23-26 were constructed. First, clone 23was constructed by cloning the region of clone 22 comprising residues164 and 165 into a clone 21 background. As shown in Table 7, changingS164I to S164V and I165 to I165V resulted in an increase in infectivity,a decrease in fusogenicity, and a decrease in PRO542 susceptibilityrelative to clone 21. Next, clone 24 was constructed by cloning theregion of clone 22 comprising positions 344 and 346 into clone 21. Asshown in Table 7, reverting the Q344R mutation to wild-type whileintroducing the A346V mutation resulted in increased infectivity,decreased fusogenicity, and decreased susceptibility to PRO542 relativeto clone 21.

Next, clone 25 was constructed by cloning the region of clone 21comprising residues 164 and 165 into a clone 22 background. As shown inTable 7, the changing from S164V and I165V to S164I and I165 resulted ina decrease in infectivity, an increase in fusogenicity, and an increasein PRO542 susceptibility relative to clone 22. Clone 26 was constructedby cloning the region of clone 21 comprising positions 164 and 165 intoclone 22. As shown in Table 7, introducing the S164I mutation andreverting the I165V mutation to wild-type resulted in decreasedinfectivity, increased fusogenicity, and increased susceptibility to PRO542 relative to clone 22.

Finally, the infectivity, fusogenicity, and PRO542 susceptibility of twoenvelope clones isolated from the same patient were assessed in the samegp120 background. The gp41 genotypes of clones 27 and 28 are presentedin Table 8, while as the infectivity, fusogenicity, and PRO542susceptibility phenotypes are presented in Table 9.

TABLE 8 536 592 601 617 621 630 633 640 668 674 683 721 833 835 clone 27M L K K K E R D S N K H V G clone 28 T F R R E Q K N N S R R L R HXB2 TL K K Q E R S S S K L V G

TABLE 9 Fusion, PRO 542 RFU % (IC50 ug/ml) Clone 27 58,149 21 0.04 Clone28 56,767 1 20.6

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TABLE 1 Cells Cell Receptor 5.25 CXCR4, CD4, CCR5 (not expressed well)BONZO 5.25.Luc4.M7 CD4, CCR5, BONZO HOS.CD4.CCR5 CD4, CCR5 HOS.CD4.CXCR4CD4,CXCR4 HOS.CD4 CD4, low level expression of CCR5 and CXCR4 HOS HT4 R5GFP wt CD4, CXCR4, CCR5 HOS.CD4.CCR5.GFP.M7#6* CD4, CXCR4, CCR5 P4.CCR5CD4, CXCR4, CCR5 U87.CD4 CD4 U87.CD4 R5 CD4, CCR5 U87.CD4 X4 CD4, CXCR4MT2 CD4, CXCR4 MT4 CD4, CXCR4 PM1 CD4, CXCR4, CCR5 CEM NKr CCR5 CD4,CXCR4, CCR5

TABLE 2 Representative viruses and reagents Viruses Envelope^(a) Source89.6, SF2 R5-X4/SI/B ARRRP^(b) 92BR014, 92US076 R5-X4/SI/B ARRRP JR-CSF,91US005 R5/NSI/B ARRRP 91US054 SI/B ARRRP NL43, MN, ELI X4/B ARRRP92HT599 X4 ARRRP 92UG031 R5/NSI/A ARRRP 92TH014, 92TH026 R5/NSI/B ARRRP92BR025, 93MW959 R5/SI/C ARRRP 92UG035 R5/NSI/D ARRRP 92TH022, 92TH023R5/NSI/E ARRRP 93BR020 R5-X4/SI/F ARRRP Antibodies Epitope SOURCE Mabs2F5, 1577 gp41 TM ARRRP Mabs IG1b12, 2G12, 17b, 48D gp120 SU ARRRPNeutralization sera #2, HIV-IG Polyclonal ARRRP Entry inhibitors TargetSource CD4-IG gp120 SU Genentech CD4-IGG2 gp120 SU Adarc SCD4 (PRO 542)Sigma Progenics T20 (DP178) gp41 TM Trimeris Rantes, MIPla/b CCR5SIGMA/ARRRP SDFla/b CXCR4 SIGMA/ARRRP AMD 3100 CXCR4 AnorMed Dextransulfate, Heparin Non-specific Sigma ^(a)R5 (CCR5 co-receptor), X4 (CXCR4co-receptor) SI (syncytium inducing), NSI (non-syncytium inducing) A, B,C, D, E, F (envelope clade designation) ^(b)AIDS Research and ReferenceReagent Program

TABLE 3 SEQ Primers Tested for the Amplification ID  of HIV Envelope NO:RT PRIMERS Primer 5′-GGA GCA TTT ACA AGC AGC AAC ACA 16 1 GC-3′ Primer5′-TTC CAG TCA VAC CTC AGG TAC-3′ 17 2 Primer5′-AGA CCA ATG ACT TAY AAG G-3′ 18 3 5′ PCR PRIMERS Primer5′-GGG CTC GAG ACC GGT CAG TGG CAA 19 4 TGA GAG TGA AG-3′ Primer5′-GGG CTC GAG ACC GGT GAG CAG AAG 20 5 ACA GTG GCA ATG A-3′ Primer5′-GGG CTC GAG ACC GGT GAG CAG AAG 21 6 ACA GTG GCA ATG-3′ 3′PCR PRIMERS Primer 5′-GGG TCT AGA ACG CGT TGC CAC CCA 22 7TCT TAT AGC AA-3′ Primer 5′-GGG TCT AGA ACG CGT CCA CTT GCC 23 8ACC CAT BTT ATA GC-3′ Primer 5′-GGG TCT AGA ACG CGT CCA CTT GCC 24 9ACC CAT BTT A-3′ Primer 5′-GAT GGT CTA AGA CGC TGT TCA ATA 25 10TCC CTG CCT AAC TC-3′

What is claimed is:
 1. A method for treating a patient having a humanimmunodeficiency virus (HIV) infection, comprising a) determining orhaving determined a length of one or more variable regions of anenvelope protein of an HIV from a patient or determining or havingdetermined a number of glycosylation sites on the envelope protein; b)comparing or having compared the length of the one or more variableregions of the envelope protein of the HIV or the number ofglycosylation sites on the envelope protein of the HIV to a length ofone or more corresponding variable regions of an envelope protein of areference HIV or a number of glycosylation sites on the envelope proteinof the reference HIV, respectively, c) determining or having determinedthat the HIV is likely to be less susceptible to a CD4 binding siteentry inhibitor than the reference HIV if the one or more variableregions of the HIV are longer than corresponding variable regions of thereference HIV or if the HIV has more glycosylation sites than thereference HIV envelope protein; and d) treating the patient with aneffective amount of the CD4 binding site entry inhibitor if the HIV isdetermined in step c) to be likely to be susceptible to the CD4 bindingsite entry inhibitor, or treating the patient with an effective amountof a different inhibitor if the HIV is determined in step c) to belikely to have reduced susceptibility to the CD4 binding site entryinhibitor.
 2. The method of claim 1, wherein the CD4 binding site entryinhibitor is PRO542 or monoclonal antibody B12.
 3. The method of claim1, wherein the reference HIV is NL4-3, HXB2, or SF2.
 4. The method ofclaim 1, wherein the HIV has at least one longer variable region thanthe reference HIV.
 5. The method of claim 1, wherein the HIV has atleast one more glycosylation site than the reference HIV.
 6. The methodof claim 1, wherein the HIV has at least one longer variable region andat least one more glycosylation site than the reference HIV.